Resolvins: bioemplates for novel therapeutic interventions

ABSTRACT

The present invention is generally drawn to novel isolated therapeutic agents, termed resolvins, generated from the interaction between a dietary omega-3 polyunsaturated fatty acid (PUFA) such as eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), cyclooxygenase-II (COX-2) and an analgesic, such as aspirin (ASA). Surprisingly, careful isolation of compounds generated from the combination of components in an appropriate environment provide di- and tri-hydroxy EPA or DHA compounds having unique structural and physiological properties. The present invention therefore provides for many new useful therapeutic di- or tri-hydroxy derivatives of EPA or DHA (resolvins) that diminish, prevent, or eliminate inflammation or PMN migration, for example. The present invention also provides methods of use, methods of preparation, and packaged pharmaceuticals for use as medicaments for the compounds disclosed throughout the specification.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation patent application of U.S. Utilitypatent application Ser. No. 10/639,714, filed Aug. 12, 2003, entitled“Resolvins: Biotemplates for Novel Therapeutic Interventions” (AttorneyDocket No. BWHI-052/US) which claims priority to U.S. ProvisionalApplication Ser. No. 60/402,798, filed on Aug. 12, 2002, entitled“Resolvins: Biotemplates for Novel Therapeutic Interventions” (AttorneyDocket No. 14448), the contents of which are incorporated herein intheir entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The work leading to this invention was supported in part by NationalInstitutes of Health (NIH) grants GM38765 and P01-DE13499. The U.S.Government therefore may have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to previously unknown therapeutic agentsderived from novel signaling and biochemical pathways that useeicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA), both ofwhich are polyunsaturated fatty acids (PUFAs) as precursors to theproduction of bioactive novel endogenous products that controlphysiologic events in inflammation and resolution in vascularendothelial reactions and neural systems (brain). More specifically, thepresent invention relates to di- and trihydroxy potent bioactiveproducts termed “Resolvins,” which are derived from the biochemicalinteractions of a 5 lipoxygenases, such as cyclooxygenase and apolyunsaturated fatty acid. In addition, therapeutic stable analogs ofresolvins that enhance their biologic properties are described that canbe used to expedite resolution by inhibiting the pro-inflammatoryamplification of leukocyte entry.

BACKGROUND OF THE INVENTION

The roles of eicosanoids in diverse physiologic and pathologic scenariosprovide clear examples of the importance of fatty acid precursors suchas arachidonic acid in cell communication, a sharp departure from theirstructural and storage assignments (3-5). Among the classes of bioactiveeicosanoids, including prostaglandins, leukotrienes (LT), lipoxins (LX)and cis-epoxyeicosatrienoic acids or EETs (4, 6), it is now apparentthat counterregulatory autacoids exist within these classes ofeicosanoids. Of the cyclooxygenase pathways, prostacyclin andthromboxane are important vascular counter-regulators (7). Ininflammation, leukotriene products of the 5-lipoxygenase arepro-inflammatory mediators (4, 8), and lipoxins generated vialipoxygenase interactions can counterregulate certainleukotriene-mediated events (for a recent review, see 9). The emergenceof temporal and spacial separation in biosynthesis of eicosanoids duringinflammation sheds light on distinct functional settings for lipoxins as“stop” or pro-resolution signals (10). Moreover, aspirin (ASA) treatmentcan pirate the lipoxin system, triggering formation of their 15-epimericor their R-containing isoform (ASA-triggered LX) that serve as LXmimetics, to mount pro-resolution status (9, 11, 12), as well asenhancers in epithelial-based anti-microbial host defense (13).

Leukocytes from several species of fish rich in omega-3 fatty acidsgenerate prostaglandins, leukotrienes and lipoxins from both arachidonicacid (C20:4) and eicosapentaenoic acid (C20:5). Their immune functionsin marine organisms appear similar to those in humans; namely, asdrivers of cell motility. Yet, fish cells generate quantitativelysimilar levels of both 4 and 5 series (EPA-derived) leukotrienes andlipoxins, which is sharply different than human tissues that usepredominantly C20:4-derived mediators (reviewed in 14). Omega-3 fattyacids such as eicosapentaenoic acid (EPA, C20:5) and docosahexaenoicacid (DHA, C22:6) may be beneficial in several human diseases includingatherosclerosis, asthma, cardiovascular, cancer (reviewed in 15), and,more recently, mental depression (16, 17) and preventing sudden deathafter myocardial infarction (18, 19). Of interest are results from theGISSI-Prevenzione trial that evaluated omega-3 polyunsaturated fattyacid supplementation with more than 11,300 patients that provideevidence for a decrease of 45% in cardiovascular death (20).

It is noteworthy that both patient groups received aspirin in the GISSItrial while comparing tocopherol vs. omega-3 supplementation (20) as dida significant number of participants in the most recent Physician Healthstudy report (18). The impact of ASA to the results of these studies wasnot tested although firmly concluding the benefits of omega-3 fattyacids in risk reduction (18, 20, 21). Eating fish rich in omega-3's isnow recommended by the American Heart Association (seehttp://www.americanheart.org). However, what is evident from animalstudies is that DHA is the bioactive cardiovascular protective componentof fish oils (22). The mechanism(s) for omega-3 protective properties inheart disease and in prostate cancer remains unclear and the molecularbases are still sought to explain the clinical phenomena associated withfish oil trials.

The heightened awareness that unresolved inflammation is important inmany chronic disorders including heart disease, atherosclerosis, asthma,and Alzheimer's disease (23, 24) leads to question whether omega-3utilization during ASA therapy is converted to endogenous bioactivecompounds relevant in human disease and health. Recently, data suggeststhat at sites of inflammation omega-3 PUFA eicosapentaenoic acid (EPA)is converted to potent bioactive products that target neutrophilrecruitment (2). Hence, COX-2, which has a larger substratetunnel/channel than COX-1 (25, 26), acts on C20:4 as well as additionalsubstrates that can be productively accommodated as exemplified by theability to convert the omega-3 polyene family of lipids (i.e., C18:3 andC20:5), possibly for tissue-specific COX-2 missions (2) such as thoseassociated with ischemic preconditioning (19), resolution (10, 12, 27)and/or other disease processes. EPA and COX-2 (2) or DHA (28-32) raisethe possibility that, in addition to arachidonic acid, omega-3 fattyacids in certain biologic processes, e.g., ischemia-induced cardiacarrhythmias (22), may serve as substrates for conversion to potentbioactive products (2). However, the biological role and significance ofproducts that could be derived from DHA in inflammation has remained tobe established.

A need therefore exists for an improved understanding of the function ofthese materials in physiology as well as the isolation of bioactiveagents that can serve to eliminate or diminish various disease states orconditions, such as those associated with inflammation.

BRIEF SUMMARY OF THE INVENTION

The present invention, in one embodiment, is drawn to isolatedtherapeutic agents generated from the interaction between a dietaryomega-3 polyunsaturated fatty acid (PUFA) such as eicosapentaenoic acid(EPA) or docosahexaenoic acid (DHA), cyclooxygenase-II (COX-2) and ananalgesic, such as aspirin (ASA). Surprisingly, careful and challengingisolation of previously unknown and unappreciated compounds aregenerated from exudates by the combination of components in anappropriate environment to provide di- and tri-hydroxy EPA or DHAcompounds having unique structural and physiological properties. Thepresent invention therefore provides for many new useful therapeutic di-or tri-hydroxy derivatives of EPA or DHA that diminish, prevent, oreliminate inflammation, for example.

The di- and tri-hydroxy EPA and DHA therapeutic agents of the inventioninclude, for example:

wherein P₁, P₂ and P₃, if present, each individually are protectinggroups, hydrogen atoms or combinations thereof;

wherein R₁, R₂ and R₃, if present, each individually are substituted orunsubstituted, branched or unbranched alkyl groups, substituted orunsubstituted aryl groups, substituted or unsubstituted, branched orunbranched alkylaryl groups, halogen atoms, hydrogen atoms orcombinations thereof;

wherein Z is —C(O)OR^(d), —C(O)NR^(c)R^(c), —C(O)H, —C(NH)NR^(c)R^(c),—C(S)H, —C(S)OR^(d), —C(S)NR^(c)R^(c), —CN;

each R^(a), if present, is independently selected from the groupconsisting of hydrogen, (C1-C6) alkyl, (C3-C8) cycloalkyl, cyclohexyl,(C4-C11) cycloalkylalkyl, (C5-C10) aryl, phenyl, (C6-C16) arylalkyl,benzyl, 2-6 membered heteroalkyl, 3-8 membered cycloheteroalkyl,morpholinyl, piperazinyl, homopiperazinyl, piperidinyl, 4-11 memberedcycloheteroalkylalkyl, 5-10 membered heteroaryl and 6-16 memberedheteroarylalkyl;

each R^(b), if present, is a suitable group independently selected fromthe group consisting of ═O, —OR^(d), (C1-C3) haloalkyloxy, —OCF₃, ═S,—SR^(d), ═NR^(d), ═NOR^(d), —NR^(c)R^(c), halogen, —CF₃, —CN, —NC, —OCN,—SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)R^(d), —S(O)₂R^(d), —S(O)₂OR^(d),—S(O)NR^(c)R^(c), —S(O)₂NR^(c)R^(c), —OS(O)R^(d), —OS(O)₂R^(d),—OS(O)₂OR^(d), —OS(O)₂NR^(c)R^(c), —C(O)R^(d), —C(O)OR^(d),—C(O)NR^(c)R^(c), —C(NH)NR^(c)R^(c), —C(NR^(a))NR^(c)R^(c),—C(NOH)R^(a), —C(NOH)NR^(c)R^(c), —OC(O)R^(d), —OC(O)OR^(d),—OC(O)NR^(c)R^(c), —OC(NH)NR^(c)R^(c), —OC(NR^(a))NR^(c)R^(c),—[NHC(O)]_(n)R^(d), —[NR^(a)C(O)]_(n)R^(d), —[NHC(O)]_(n)OR^(d),—[NR^(a)C(O)]_(n)OR^(d), —[NHC(O)]_(n)NR^(c)R^(c),—[NR^(a)C(O)]_(n)NR^(c)R^(c), —[NHC(NH)]_(n)NR^(c)R^(c) and—[NR^(a)C(NR^(a))]_(n)NR^(c)R^(c);

each R^(c), if present, is independently a protecting group or R^(a),or, alternatively, each R^(c) is taken together with the nitrogen atomto which it is bonded to form a 5 to 8-membered cycloheteroalkyl orheteroaryl which may optionally include one or more of the same ordifferent additional heteroatoms and which may optionally be substitutedwith one or more of the same or different R^(a) or suitable R^(b)groups;

each n, independently, if present, is an integer from 0 to 3;

each R^(d), independently, if present, is a protecting group or R^(a);

in particular, Z is a carboxylic acid, ester, amide, thiocarbamate,carbamate, thioester, thiocarboxamide or a nitrile;

wherein X, if present, is a substituted or unsubstituted methylene, anoxygen atom, a substituted or unsubstituted nitrogen atom, or a sulfuratom;

wherein Q represents one or more substituents and each Q individually,if present, is a halogen atom or a branched or unbranched, substitutedor unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkoxy,aryloxy, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl,amino, hydroxy, cyano, carboxyl, alkoxycarbonyloxy, aryloxycarbonyloxyor aminocarbonyl group;

wherein U is a branched or unbranched, substituted or unsubstitutedalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkoxy, aryloxy,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl,alkoxycarbonyloxy, and aryloxycarbonyloxy group;

and pharmaceutically acceptable salts thereof.

In certain embodiments, P₁, P₂, and P₃, if present, each individuallyare hydrogen atoms and Z is a carboxylic acid or ester. In otherembodiments, X is an oxygen atom, one or more P's are hydrogen atoms, Zis a carboxylic acid or ester. In still other embodiments, Q is one ormore halogen atoms, one or more P's are hydrogen atoms, and Z is acarboxylic acid or ester.

In certain embodiments, R₁, R₂ and R₃, if present, are each individuallylower alkyl groups, such as methyl, ethyl, and propyl and can behalogenated, such as trifluoromethyl. In one aspect, at least one of R₁,R₂ and R₃, if present, is not a hydrogen atom. Generally, Z is acarboxylic acid and one or more P's are hydrogen atoms.

In certain embodiments, when OP₃ is disposed terminally within theresolvin analog, the protecting group can be removed to afford ahydroxyl. Alternatively, in certain embodiments, the designation of OP₃serves to denote that the terminal carbon is substituted with one ormore halogens, i.e., the terminal C-18, C-20, or C-22 carbon, is atrifluoromethyl group, or arylated with an aryl group that can besubstituted or unsubstituted as described herein. Such manipulation atthe terminal carbon serves to protect the resolvin analog from omegaP₄₅₀ metabolism which can lead to biochemical inactivation.

In one aspect, the resolvins described herein that contain epoxide,cyclopropane, azine, or thioazine rings within the structure also serveas enzyme inhibitors that increase endogenous resolvin levels in vivoand block “pro” inflammatory substances, their formation and action invivo, such as leukotrienes and/or LTB₄.

Another embodiment of the present invention is directed topharmaceutical compositions of the novel compounds described throughoutthe specification.

The present invention also provides methods to treat various diseasestates and conditions, including for example, inflammation.

The present invention further provides various methods to prepare thenovel compounds described throughout the specification.

The present invention also provides packaged pharmaceuticals thatcontain the novel di- and tri-hydroxy EPA and DHA compounds describedthroughout the specification for use in treatment with various diseasestates and conditions.

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description. As will be apparent, the inventionis capable of modifications in various obvious aspects, all withoutdeparting from the spirit and scope of the present invention.Accordingly, the drawings and detailed description are to be regarded asillustrative in nature and not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts inflammatory exudates from mice treated with ASA togenerate novel compounds: LC-MS-MS-based lipodomic analysis. Panel A:TNFα-induced leukocyte exudates from dorsal air pouches. Samples werecollected at 6 h from FVB mice given ASA and DHA (See Methods). Selectedion chromatogram (m/z 343) showing the production of 17R-HDHA, 7S-HDHA,and 4S-HDHA. Using the diene UV chromophores for quantitation, 7-HDHAwas ˜15% of the exudate materials and was identified using a SIM tracefor m/z 141 with ms/ms 343. In some exudates 17S-HDHA was also presentfrom lipoxygenase-dependent routes.

MS-MS for Panel B: 17R-HDHA (m/z 343); Panel C: 7S,17R-diHDHA (m/z 359);and Panel D: 4,11,17R-triHDHA (m/z 375). See below for diagnostic ions.Results are representative of n=7.

FIG. 2 depicts novel ASA triggered HDHA products generated by humanrecombinant COX-2-aspirin: 17R-HDHA. Human recombinant COX-2 treated inthe presence and absence of ASA (2 mM) was incubated with DHA (10 μM, 30min, 37° C.). Incubations were stopped with 2 ml cold methanol,extracted and taken for LC-MS-MS analyses. Results are representative ofincubations from >8 separate experiments, some with 1-¹⁴C-labeled DHA.Upper Panels: LC-MS-MS chromatogram of m/z 343 showing the presence ofmono-HDHA. Lower: MS-MS spectrum of (left) 13-HDHA without ASA treatmentand (right) 17R-HDHA with ASA treatment.

FIG. 3 depicts endogenous 17R-HDHA from brain and human microglial cellstreated with aspirin. A) LC-MS-MS chromatogram obtained from brain forrelative abundance at m/z 327 for DHA and m/z 343 for the monohydroxyproduct. B) MS-MS spectrum of brain 17R-HDHA (m/z 343). Murine brainsamples were incubated with ASA (45 min, 37° C.). Results arerepresentative of n=6 mice treated with ASA vs. 5 mice without ASA. C)Human microglial cells (HMG) treated with ASA; MS-MS spectrum of HMG17R-HDHA. 10×10⁶ cells were exposed to TNFa (50 ng/ml) and incubated (24h, 37° C.). Cells were treated with ASA (500 μM, 30 min, 37° C.)followed by addition of ionophore A23187 (5μ, 25-30 min). Incubationswere stopped with MeOH, extracted and analyzed by tandem UV, LC-MS-MS(FIG. 3 inset shows UV-chromatogram plotted at 235 nm absorbance) (n=4,d=20). Both 17R-HDHA and DHA were identified on the basis of individualretention times, parent ions, and daughter ions obtained.

FIG. 4 depicts hypoxic HUVECs treated with ASA generate 17R-HDHA. HUVECswere exposed to TNFα and IL-1β (both 1 ng/ml) and placed in a hypoxiachamber (3 h). The cells were treated with ASA (500 μM, 30 min) followedby DHA (20 μg/10⁶ cells/10 ml plate) and A23187 (2 μM, 60 min). Panel A:LC-MS-MS chromatogram of ion m/z 343 shows the presence of 17R-HDHA.Panel B: MS-MS spectrum (RT=21.2 min) of 17R-HDHA identified byretention time, parent ions, and daughter ions and matched withproperties and authentic NMR qualified standard.

FIG. 5 depicts Bioimpact properties of omega-3-derived Resolvins. A)Human glioma cells: Inhibition of TNF-stimulated IL-1β transcriptsDBTRG-05MG cells 10⁶/ml were stimulated with 50 ng/ml of humanrecombinant TNFα for 16 hours to induce expression of IL-1β transcripts.Concentration dependence with COX-2 products: 17-HDHA (▪), 13-HDHA ()and di-/tri-HDHA (□). The IC₅₀ for both compounds is ˜50 pM. (insets)Results are representative of RT-PCR gels of MG cells exposed to 100 nMof 13-HDHA or 17-HDHA and graphed after normalization of the IL-1βtranscripts using GAPDH. B) Influence of eicosanoids and docosanoids onfMLP-induced neutrophil migration across micovascular endothelialmonolayers Neutrophils (1×10⁶ cells/monolayer) were exposed to vehiclecontaining buffer, or indicated concentrations of aspirin-triggered LXA₄analog (closed diamonds) 5S,12,18R-triHEPE (closed squares), 17R-HDHA(closed circles) or 13-HDHA (closed triangles) for 15 minutes at 37° C.Neutrophils were then layered on HMVEC monolayers and stimulated totransmigrate by a 10⁻⁸ M fMLP gradient for 1 hr at 37° C. Transmigrationwas assessed by quantitation of the neutrophil marker myeloperoxidase.Results are presented as mean±SEM number of PMN (n=8-12 monolayers percondition). C) Reduction of PMN in murine peritonitis and skin pouchperitonitis: Compounds (100 ng in 120 μL sterile saline) were injectedby intravenous bolus injection into the mouse tail vein and followed by1 ml zymosan A (1 mg/ml) into the peritoneum. Peritoneal lavages werecollected (2 h) and cell types were enumerated.

Air pouch—Compounds (dissolved in 500 μL of PBS without Ca²⁺ or Mg²⁺)injected into the air pouch via intrapouch injection or via intravenousadministration (in 120 μl sterile saline) followed by intrapouchinjection of TNFα. Four hours later air pouch lavages were collected andcells were enumerated and differentiated. Compounds were prepared bybiogenic synthesis or isolated from in vivo exudates. The ratio of7,17R-diHDHA to 4,17R-diHDHA was ˜8:1; the ratio of 4,11,17R-triHDHA and7,16,17R-triHDHA was ˜2:1; and the ratio of di to triHDHA was ˜1:1.3.Exudate transfers to a native mouse (described herein). ATLa denotes15-epi-16-para(fluoro)-phenoxy-LXA₄ (administered at 100 ng/mouse).Values represent mean+/−SEM from 3-4 different mice; *P<0.05 wheninfiltrated PMN is compared to vehicle control.

FIG. 6 depicts resolvin production by human PMNs exposed to microbialzymosan: novel 17R di- and triHDHA. Human PMNs (50×10⁶ cells/ml)incubated with zymosan A (100 ng/ml) and 17R-HDHA (5 μg/ml, 40 min, 37°C.). Results are representative of n=4.

FIG. 7 depicts that inflammatory exudate produces 17R-containing di- andtrihydroxy tetraenes and triene-containing compounds: LC-MS-MS. See FIG.1 for details. Exudates were obtained and analyzed by proceduresessentially identical to those described in FIG. 1. Panel A: m/z wereplotted at 375 (upper), 359 (middle), and 343 (lower). Panel B: UVabsorbance was plotted at 300 nm to mark tetraene-containingchromatophores. Panel C: MS-MS of 7S,8,17R-triHDHA.

FIG. 8 depicts a biosynthetic scheme proposed for resolvins: aspirintriggered omega-3-derived products. Acetylation of COX-2 by ASAtreatment generates novel 17R-H(p)DHA from DHA that is reduced to itscorresponding alcohol and converted via sequential actions of aleukocyte 5-lipoxygenase and leads to formation of both dihydroxy- andtrihydroxy-containing docosanoids that retain their 17R configuration.Pathways are denoted for omega oxidation products that are likely to bein vivo markers of enzymatic inactivation. The resolvin pathways appearto be maximally induced during the “spontaneous resolution” phase ofinflammation and compounds are activated to dampen PMN infiltration,which reduces exudate PMN numbers to promote pro-resolution ofinflammatory (Resolvins from EPA, the 18R-HEPE series, are denoted) thatleads to potent inhibitors of PMN recruitment in vitro and in vivo (seepathway, right, text and Ref. 2). The complete stereochemistries of thenew di- and trihydroxy-containing compounds remain to be established andare depicted here in their likely configuration based on biogenic totalsynthesis. See Table 2 and text for further details.

FIG. 9 provides a lipodomics based analysis/flow diagram for theapproach to isolate and characterize the complex and unknown compoundsof the present invention.

FIG. 10 provides a depiction of the general metabolic pathway in whichproduction of resolvin di and tri-hydroxy compounds are produced.

FIG. 11 is a more detailed illustration which depicts the production ofresolving di and tri-hydroxy compounds from HDA or EPA from a PUFA andaspirin within inflamed tissue.

FIG. 12 provides a schematic depiction of some of the di and tri-HDHAcompounds of the invention.

FIG. 13 shows a biochemical pathway/conversion of EPA with COX-2 to formdi and tri-hydroxy EPA compounds.

FIG. 14 is another schematic depiction of a biochemicalpathway/conversion of DHA with COX-2 to form di and tri-hydroxy DHAcompounds.

FIG. 15 shows the physical properties for 5S,18(+/−)-diHEPE.

FIG. 16 is a direction comparison of 5S,18(+/−)-diHEPE and Resolvin E1,demonstrating a reduction of neutrophil infiltration in Zymosan-inducedperitonitis.

FIG. 17 depicts a docosatriene pathway.

FIG. 18 is a comparison of 4,17S-diHDHA and 10,17S docosatriene inreduction of leukocyte infiltration in Zymosan-induced peritonitis.

DETAILED DESCRIPTION

The present invention provides methods for preparing novelanti-inflammatory agents and discloses the structures of novelendogenously generated anti-inflammatory mediators that are generated inresolution. The invention is based on the structural elucidation ofseveral new classes of compounds that are generated in vivo duringinflammation, which are termed “resolvins.” The structural elucidationof the compounds and the mechanisms of their biosynthesis at sites ofinflammation in vivo in murine systems via vascular leukocyteinteractions and in brain when aspirin is taken are presented throughoutthe specification. This structural elucidation of novel biochemicalpathways and compounds that serve as endogenous mediators inanti-inflammation and/or pro-resolution forms the basis of a novelapproach to active anti-inflammatories that expedites resolution.

From structural elucidation, these novel compounds are “activeingredients” that the body converts via novel biochemical pathways toendogenous omega-3 fatty acid-derived mediators that haveanti-inflammatory properties that we've uncovered in murine models.These results provide that these compounds, when generated in vivo inhumans, are responsible, at least in part, for the beneficial actions ofeating fish and aspirin therapy.

The structural elucidation of these pathways, biological properties andstructural elucidation of novel compounds formulates the basis for anovel therapeutic approach, namely administering these compounds and/orrelated structures/analogs with greater biostability and chemicalstability as new therapeutic approaches to expedite resolution and evokeanti-inflammation status.

Along these lines, the new structures, pathways, and examples of novelchemical classes of analogs based on these natural resolvin compoundsare presented in the illustrations and figures throughout thisspecification. Most importantly, with the description of these novelpathways and physical properties of the resolvins, one claim can bedirected for assaying these compounds in human fluids (blood, urine,breast milk), biopsied material, etc. as treatment markers to gaugeeffective n-3 status levels as indices for developing a therapeuticbasis for anti-inflammation. This includes LC-MS-MS and GC-MS propertiesand could also lead to the development of much easier to handle ELISAassays monitoring these novel products.

Aspirin is unique among current therapies because it acetylatescyclooxygenase-2 (COX-2) enabling the biosynthesis of R-containingprecursors of endogenous anti-inflammatory and pro-resolving mediators(1, 2). The present invention provides that lipidomic analysis ofTNFα-induced exudates obtained in the resolution phase from mice treatedwith aspirin (ASA) and docosahexaenoic acid (DHA; C22:6) produce a novelfamily of bioactive 17R-hydroxy-containing di-, andtrihydroxy-docosanoids via COX-2 initiated pathways. Murine braintreated with aspirin produced 17R-hydroxydocosahexaenoic acid (17R-HDHA)from endogenous sources as did cytokine activated human microglialcells.

Human recombinant COX-2 converted DHA to 13-hydroxy-DHA that switchedwith ASA treatment to 17R-HDHA, which proved to be a major route inhypoxic human vascular endothelial cells that express COX-2. Humanneutrophils engaged in phagocytosis transformed COX-2-ASA-derived17R-hydroxy-DHA into two sets of novel 17R-hydroxy retaining di- andtrihydroxy products; one initiated via oxygenation at carbon 7 and theother at carbon 4 that generates epoxide intermediates. COX-2-ASAgenerated docosanoids (i.e., 17R-HDHA) inhibited cytokine expression(IC₅₀˜50 pM) by microglial cells. In both murine dermal inflammation andperitonitis, the 17R series compounds at ng doses (e.g.4S,11,17R-triHDHA, 7S,8,17R-triHDHA, and 7S,17R-diHDHA) reduced 40-80%leukocytic exudates.

These results indicate that COX-2-bearing murine and human cells (i.e.neural, vascular, leukocytes and exudates) with aspirin treatmentconvert DHA to novel 17R-hydroxy-containing series of docosanoids thatare potent regulators in acute inflammation-resolution. These redundantbiosynthetic pathways utilize omega-3 fatty acids during multi-cellularevents in resolution to produce endogenous protective compounds (termedResolvins) that enhance pro-resolution status. Moreover, these resultscan provide a molecular rationale for the utilization of omega-3 DHA andaspirin as well as omega-3 fatty acid dietary supplementation in chronicinflammatory diseases, neoplasia, and cardiovascular disease.

The present invention provides that aspirin treatment of murine in vivoand human tissues in vitro carrying COX-2 initiates the production ofnovel 17R-hydroxy series docosanoids via previously undescribedbiosynthetic circuits that counterregulate pro-inflammatory responses(i.e., cytokine production, peritonitis). During stress, these cellularpathways utilize omega-3 fatty acids to biosynthesize endogenouscompounds that serve in anti-inflammation signaling. Thus, the newfamily of compounds are referred to as Resolvins because they are i)generated during the resolution phase and ii) chemically redundantsignals that play protective roles in dampening inflammation to promotea pro-resolution status.

The present invention is drawn to methods for treating or preventinginflammation in a subject by administration of a combination of apolyunsaturated fatty acid(s) (PUFA(s)) and aspirin, i.e.,polyunsaturated fatty acids including C20:5 and C22:6. In oneembodiment, the omega fatty acid, e.g., C20:3 or C22:6, and ananalgesic, such as aspirin, are administered at two different times.

The phrase “resolvin mediated interaction” is intended to includedisease states or conditions caused by or associated with one or moretypes of inflammation associated with cytokine, leukocyte or PMNregulation and regulation by one or more of the therapeutic analogsdescribed throughout the specification for the pharmacologic inhibitonof inflammatory diseases, vascular disorders and neuronal inflammation.In one embodiment, the disease state includes, for example, thosediseases that afflict a subject by associating with or interfering withcytokine, leukocyte or PMN regulation within the subject. Such diseasestates or conditions are described throughout the specification, videinfra, and are incorporated herein in their entirety. Presently unknownconditions related to cytokine, leukocyte or PMN regulation that may bediscovered in the future are encompassed by the present invention, sincethe characterization as conditions related to resolvin mediatedinteraction(s) will be readily determinable by persons skilled in theart.

Resolvins are natural counter regulatory lipid mediators in host defensemechanisms that protect host tissues from effector cell mediated injuryand over amplification of acute inflammation to dampen the inflammatoryresponse, i.e., counterregulative. Some known chronic inflammatorydiseases may represent the loss of and/or genetically program lowresolvin edogenous responders and/or levels. The resolvin analogsdescribed throughout the specification can be used to replace, enhanceand/or treat the loss of these substances therapeutically and therbypharmacologically resolve inflammation by inhibiting leukocyterecruitment and amplification, namely inhibition of the amplification ofinflammation.

The present invention is also drawn to methods for treating arterialinflammation, arthritis, psoriasis, urticara, vasculitis, asthma, ocularinflammation, pulmonary inflammation, pulmonary fibrosis, seborrheicdermatitis, pustular dermatosis, or cardiovascular diseases in a subjectby administration of a combination of an omega fatty acid and ananalgesice, such as aspirin to the subject. Disease states or conditionsthat are associated with inflammation (hence “resolving”), therecruitment of neutrophils, leukocytes and/or cytokines are includedwithin the general scope of inflammation and include, for example,Addiction, AIDS, Alcohol-related disorders, Allergy, Alzheimer'sdisease, Anesthesiology, Anti-infectives, Anti-inflammatory agents,Arthritis, Asthma, Atherosclerosis, Bone diseases, Breast cancer,Cancer, Cardiovascular diseases, Child heath, Colon cancer, Congenitaldefects, Decision analysis, Degenerative neurologic disorders, Dementia,Dermatology, Diabetes mellitus, Diagnostics, Drug delivery, Drugdiscovery/screen, Endocrine disorders, ENT, Epidemiology, Eye diseases,Fetal and maternal medicine, Gastrointestinal disorders, Gene therapy,Genetic diagnostics, Genetics, Genitourinary disorders, Geriatricmedicine, Growth and Development, Hearing, Hematologic disorders,Hepatobiliary disorders, Hypertension, Imaging, Immunology, Infectiousdiseases, Leukemia/lymphoma, Lung cancer, Metabolic disorders,Neonatology, Neurological disorders, Neromuscular disorders, Nuclearmedicine, Obesity/eating disorders, Orthopedic, Other, Parasiticdiseases, Perinatal disorders, Pregnancy, Preventative medicine,Prostate cancer, Psychiatric disorders, Pulmonary disorders, Radiology,Renal disorders, Reproduction, Rheumatic diseases, Stroke, Surgical,Transplantation, Vaccines, Vascular medicine, Wound healing, oralinfections, periodontal disease, brain injury, trauma and neuronalinflammation, and Women's health.

Terms and abbreviations used throughout the specification include:

ASA, aspirinCOX, cyclooxygenaseEPA, eicosapentaenoic acidDHA, docosahexaenoic acidGC-MS, gas chromatography-mass spectrometry4S-HDHA, 4S-hydroxy-5E,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid17S-HDHA, 17S-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid17R/S-HDHA, 17R/S-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid17R-HDHA, 17-hydroxy-docosa-4Z,7Z,10Z,13Z,15E,19Z-hexaenoic acid7S,17R-dihydroxy-DHA,7S,17R-dihydroxy-docosa-4Z,8E,10Z,13Z,15E,19Z-hexaenoic acid4S,17R-dihydroxy-DHA,4S-17R-dihydroxy-docosa-5E,7Z,10Z,13Z,15E,19Z-hexaenoic acid7S,17R,22-trihydroxy-DHA,7S,17R,22-trihydroxy-docosa-4Z,8Z,10Z,13Z,15E,19Z-hexaenoic acid4S,11,17R-trihydroxy-DHA,4S,11,17S,-trihydroxy-docosa-5E,7E,9Z,13Z,15E,19Z-hexaenoic acidLC-UV-MS-MS, liquid chromatography-UV diode array detector-tandem massspectrometryLO, lipoxygenaseLT, leukotrieneLX, lipoxinsPDA, photodiode array detectorPUFA, polyunsaturated fatty acids

“Alkyl” by itself or as part of another substituent refers to asaturated or unsaturated branched, straight-chain or cyclic monovalenthydrocarbon radical having the stated number of carbon atoms (i.e.,C1-C6 means one to six carbon atoms) that is derived by the removal ofone hydrogen atom from a single carbon atom of a parent alkane, alkeneor alkyne. Typical alkyl groups include, but are not limited to, methyl;ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl,prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Wherespecific levels of saturation are intended, the nomenclature “alkanyl,”“alkenyl” and/or “alkynyl” is used, as defined below. In preferredembodiments, the alkyl groups are (C1-C6) alkyl.

“Alkanyl” by itself or as part of another substituent refers to asaturated branched, straight-chain or cyclic alkyl derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkane. Typical alkanyl groups include, but are not limited to,methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl(isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl,butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl),2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like. Inpreferred embodiments, the alkanyl groups are (C1-C6) alkanyl.

“Alkenyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon double bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkene. The group may be in eitherthe cis or trans conformation about the double bond(s). Typical alkenylgroups include, but are not limited to, ethenyl; propenyls such asprop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl,cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such asbut-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.;and the like. In preferred embodiments, the alkenyl group is (C2-C6)alkenyl.

“Alkynyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon triple bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkyne. Typical alkynyl groupsinclude, but are not limited to, ethynyl; propynyls such asprop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. In preferredembodiments, the alkynyl group is (C2-C6) alkynyl.

“Alkyldiyl” by itself or as part of another substituent refers to asaturated or unsaturated, branched, straight-chain or cyclic divalenthydrocarbon group having the stated number of carbon atoms (i.e., C1-C6means from one to six carbon atoms) derived by the removal of onehydrogen atom from each of two different carbon atoms of a parentalkane, alkene or alkyne, or by the removal of two hydrogen atoms from asingle carbon atom of a parent alkane, alkene or alkyne. The twomonovalent radical centers or each valency of the divalent radicalcenter can form bonds with the same or different atoms. Typicalalkyldiyl groups include, but are not limited to, methandiyl; ethyldiylssuch as ethan-1,1-diyl, ethan-1,2-diyl, ethen-1,1-diyl, ethen-1,2-diyl;propyldiyls such as propan-1,1-diyl, propan-1,2-diyl, propan-2,2-diyl,propan-1,3-diyl, cyclopropan-1,1-diyl, cyclopropan-1,2-diyl,prop-1-en-1,1-diyl, prop-1-en-1,2-diyl, prop-2-en-1,2-diyl,prop-1-en-1,3-diyl, cycloprop-1-en-1,2-diyl, cycloprop-2-en-1,2-diyl,cycloprop-2-en-1,1-diyl, prop-1-yn-1,3-diyl, etc.; butyldiyls such as,butan-1,1-diyl, butan-1,2-diyl, butan-1,3-diyl, butan-1,4-diyl,butan-2,2-diyl, 2-methyl-propan-1,1-diyl, 2-methyl-propan-1,2-diyl,cyclobutan-1,1-diyl; cyclobutan-1,2-diyl, cyclobutan-1,3-diyl,but-1-en-1,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3-diyl,but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1-diyl,2-methanylidene-propan-1,1-diyl, buta-1,3-dien-1,1-diyl,buta-1,3-dien-1,2-diyl, buta-1,3-dien-1,3-diyl, buta-1,3-dien-1,4-diyl,cyclobut-1-en-1,2-diyl, cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl,cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl,but-1-yn-1,3-diyl, but-1-yn-1,4-diyl, buta-1,3-diyn-1,4-diyl, etc.; andthe like. Where specific levels of saturation are intended, thenomenclature alkanyldiyl, alkenyldiyl and/or alkynyldiyl is used. Whereit is specifically intended that the two valencies are on the samecarbon atom, the nomenclature “alkylidene” is used. In preferredembodiments, the alkyldiyl group is (C1-C6) alkyldiyl. Also preferredare saturated acyclic alkanyldiyl groups in which the radical centersare at the terminal carbons, e.g., methandiyl (methano);ethan-1,2-diyl(ethano); propan-1,3-diyl (propano); butan-1,4-diyl(butano); and the like (also referred to as alkylenos, defined infra).

“Alkyleno” by itself or as part of another substituent refers to astraight-chain saturated or unsaturated alkyldiyl group having twoterminal monovalent radical centers derived by the removal of onehydrogen atom from each of the two terminal carbon atoms ofstraight-chain parent alkane, alkene or alkyne. The locant of a doublebond or triple bond, if present, in a particular alkyleno is indicatedin square brackets. Typical alkyleno groups include, but are not limitedto, methano; ethylenos such as ethano, etheno, ethyno; propylenos suchas propano, prop[1]eno, propa[1,2]dieno, prop[1]yno, etc.; butylenossuch as butano, but[1]eno, but[2]eno, buta[1,3]dieno, but[1]yno,but[2]yno, buta[1,3]diyno, etc.; and the like. Where specific levels ofsaturation are intended, the nomenclature alkano, alkeno and/or alkynois used. In preferred embodiments, the alkyleno group is (C1-C6) or(C1-C3) alkyleno. Also preferred are straight-chain saturated alkanogroups, e.g., methano, ethano, propano, butano, and the like.

“Heteroalkyl,” Heteroalkanyl,” Heteroalkenyl,” Heteroalkynyl,”Heteroalkyldiyl” and “Heteroalkyleno” by themselves or as part ofanother substituent refer to alkyl, alkanyl, alkenyl, alkynyl, alkyldiyland alkyleno groups, respectively, in which one or more of the carbonatoms are each independently replaced with the same or differentheteratoms or heteroatomic groups. Typical heteroatoms and/orheteroatomic groups which can replace the carbon atoms include, but arenot limited to, —O—, —S—, —S—O—, —NR′—, —PH—, —S(O)—, —S(O)₂—,—S(O)NR′—, —S(O)₂NR′—, and the like, including combinations thereof,where each R′ is independently hydrogen or (C1-C6) alkyl.

“Cycloalkyl” and “Heterocycloalkyl” by themselves or as part of anothersubstituent refer to cyclic versions of “alkyl” and “heteroalkyl”groups, respectively. For heteroalkyl groups, a heteroatom can occupythe position that is attached to the remainder of the molecule. Typicalcycloalkyl groups include, but are not limited to, cyclopropyl;cyclobutyls such as cyclobutanyl and cyclobutenyl; cyclopentyls such ascyclopentanyl and cyclopentenyl; cyclohexyls such as cyclohexanyl andcyclohexenyl; and the like. Typical heterocycloalkyl groups include, butare not limited to, tetrahydrofuranyl (e.g., tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, etc.), piperidinyl (e.g., piperidin-1-yl,piperidin-2-yl, etc.), morpholinyl (e.g., morpholin-3-yl,morpholin-4-yl, etc.), piperazinyl (e.g., piperazin-1-yl,piperazin-2-yl, etc.), and the like.

“Acyclic Heteroatomic Bridge” refers to a divalent bridge in which thebackbone atoms are exclusively heteroatoms and/or heteroatomic groups.Typical acyclic heteroatomic bridges include, but are not limited to,—O—, —S—, —S—O—, —NR′—, —PH—, —S(O)—, —S(O)₂—, —S(O)NR′—, —S(O)₂NR′—,and the like, including combinations thereof, where each R′ isindependently hydrogen or (C1-C6) alkyl.

“Parent Aromatic Ring System” refers to an unsaturated cyclic orpolycyclic ring system having a conjugated π electron system.Specifically included within the definition of “parent aromatic ringsystem” are fused ring systems in which one or more of the rings arearomatic and one or more of the rings are saturated or unsaturated, suchas, for example, fluorene, indane, indene, phenalene,tetrahydronaphthalene, etc. Typical parent aromatic ring systemsinclude, but are not limited to, aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexylene, indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, tetrahydronaphthalene, triphenylene, trinaphthalene, and thelike, as well as the various hydro isomers thereof.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon group having the stated number of carbonatoms (i.e., C5-C15 means from 5 to 15 carbon atoms) derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexylene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like, as well as thevarious hydro isomers thereof. In preferred embodiments, the aryl groupis (C5-C15) aryl, with (C5-C10) being even more preferred. Particularlypreferred aryls are cyclopentadienyl, phenyl and naphthyl.

“Arylaryl” by itself or as part of another substituent refers to amonovalent hydrocarbon group derived by the removal of one hydrogen atomfrom a single carbon atom of a ring system in which two or moreidentical or non-identical parent aromatic ring systems are joineddirectly together by a single bond, where the number of such direct ringjunctions is one less than the number of parent aromatic ring systemsinvolved. Typical arylaryl groups include, but are not limited to,biphenyl, triphenyl, phenyl-naphthyl, binaphthyl, biphenyl-naphthyl, andthe like. Where the number of carbon atoms in an arylaryl group arespecified, the numbers refer to the carbon atoms comprising each parentaromatic ring. For example, (C5-C15) arylaryl is an arylaryl group inwhich each aromatic ring comprises from 5 to 15 carbons, e.g., biphenyl,triphenyl, binaphthyl, phenylnaphthyl, etc. Preferably, each parentaromatic ring system of an arylaryl group is independently a (C5-C15)aromatic, more preferably a (C5-C10) aromatic. Also preferred arearylaryl groups in which all of the parent aromatic ring systems areidentical, e.g., biphenyl, triphenyl, binaphthyl, trinaphthyl, etc.

“Biaryl” by itself or as part of another substituent refers to anarylaryl group having two identical parent aromatic systems joineddirectly together by a single bond. Typical biaryl groups include, butare not limited to, biphenyl, binaphthyl, bianthracyl, and the like.Preferably, the aromatic ring systems are (C5-C15) aromatic rings, morepreferably (C5-C10) aromatic rings. A particularly preferred biarylgroup is biphenyl.

“Arylalkyl” by itself or as part of another substituent refers to anacyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced withan aryl group. Typical arylalkyl groups include, but are not limited to,benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. Where specific alkyl moietiesare intended, the nomenclature arylalkanyl, arylakenyl and/orarylalkynyl is used. In preferred embodiments, the arylalkyl group is(C6-C21) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of thearylalkyl group is (C1-C6) and the aryl moiety is (C5-C15). Inparticularly preferred embodiments the arylalkyl group is (C6-C13),e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is(C1-C3) and the aryl moiety is (C5-C10).

“Parent Heteroaromatic Ring System” refers to a parent aromatic ringsystem in which one or more carbon atoms are each independently replacedwith the same or different heteroatoms or heteroatomic groups. Typicalheteroatoms or heteroatomic groups to replace the carbon atoms include,but are not limited to, N, NH, P, O, S, S(O), S(O)₂, Si, etc.Specifically included within the definition of “parent heteroaromaticring systems” are fused ring systems in which one or more of the ringsare aromatic and one or more of the rings are saturated or unsaturated,such as, for example, benzodioxan, benzofuran, chromane, chromene,indole, indoline, xanthene, etc. Also included in the definition of“parent heteroaromatic ring system” are those recognized rings thatinclude common substituents, such as, for example, benzopyrone and1-methyl-1,2,3,4-tetrazole. Typical parent heteroaromatic ring systemsinclude, but are not limited to, acridine, benzimidazole, benzisoxazole,benzodioxan, benzodioxole, benzofuran, benzopyrone, benzothiadiazole,benzothiazole, benzotriazole, benzoxaxine, benzoxazole, benzoxazoline,carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole,indazole, indole, indoline, indolizine, isobenzofuran, isochromene,isoindole, isoindoline, isoquinoline, isothiazole, isoxazole,naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine,phenanthroline, phenazine, phthalazine, pteridine, purine, pyran,pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole,pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike.

“Heteroaryl” by itself or as part of another substituent refers to amonovalent heteroaromatic group having the stated number of ring atoms(e.g., “5-14 membered” means from 5 to 14 ring atoms) derived by theremoval of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, benzimidazole,benzisoxazole, benzodioxan, benzodiaxole, benzofuran, benzopyrone,benzothiadiazole, benzothiazole, benzotriazole, benzoxazine,benzoxazole, benzoxazoline, carbazole, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike, as well as the various hydro isomers thereof. In preferredembodiments, the heteroaryl group is a 5-14 membered heteroaryl, with5-10 membered heteroaryl being particularly preferred.

“Heteroaryl-Heteroaryl” by itself or as part of another substituentrefers to a monovalent heteroaromatic group derived by the removal ofone hydrogen atom from a single atom of a ring system in which two ormore identical or non-identical parent heteroaromatic ring systems arejoined directly together by a single bond, where the number of suchdirect ring junctions is one less than the number of parentheteroaromatic ring systems involved. Typical heteroaryl-heteroarylgroups include, but are not limited to, bipyridyl, tripyridyl,pyridylpurinyl, bipurinyl, etc. Where the number of atoms are specified,the numbers refer to the number of atoms comprising each parentheteroaromatic ring systems. For example, 5-15 memberedheteroaryl-heteroaryl is a heteroaryl-heteroaryl group in which eachparent heteroaromatic ring system comprises from 5 to 15 atoms, e.g.,bipyridyl, tripuridyl, etc. Preferably, each parent heteroaromatic ringsystem is independently a 5-15 membered heteroaromatic, more preferablya 5-10 membered heteroaromatic. Also preferred are heteroaryl-heteroarylgroups in which all of the parent heteroaromatic ring systems areidentical.

“Biheteroaryl” by itself or as part of another substituent refers to aheteroaryl-heteroaryl group having two identical parent heteroaromaticring systems joined directly together by a single bond. Typicalbiheteroaryl groups include, but are not limited to, bipyridyl,bipurinyl, biquinolinyl, and the like. Preferably, the heteroaromaticring systems are 5-15 membered heteroaromatic rings, more preferably5-10 membered heteroaromatic rings.

“Heteroarylalkyl” by itself or as part of another substituent refers toan acyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced with aheteroaryl group. Where specific alkyl moieties are intended, thenomenclature heteroarylalkanyl, heteroarylakenyl and/orheteroarylalkynyl is used. In preferred embodiments, the heteroarylalkylgroup is a 6-21 membered heteroarylalkyl, e.g., the alkanyl, alkenyl oralkynyl moiety of the heteroarylalkyl is (C1-C6) alkyl and theheteroaryl moiety is a 5-15-membered heteroaryl. In particularlypreferred embodiments, the heteroarylalkyl is a 6-13 memberedheteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety is (C1-C3)alkyl and the heteroaryl moiety is a 5-10 membered heteroaryl.

“Halogen” or “Halo” by themselves or as part of another substituent,unless otherwise stated, refer to fluoro, chloro, bromo and iodo.

“Haloalkyl” by itself or as part of another substituent refers to analkyl group in which one or more of the hydrogen atoms is replaced witha halogen. Thus, the term “haloalkyl” is meant to includemonohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls.For example, the expression “(C1-C2) haloalkyl” includes fluoromethyl,difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl,1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, etc.

The above-defined groups may include prefixes and/or suffixes that arecommonly used in the art to create additional well-recognizedsubstituent groups. As examples, “alkyloxy” or “alkoxy” refers to agroup of the formula —OR^(d), “alkylamine” refers to a group of theformula —NHR″ and “dialkylamine” refers to a group of the formula—NR″R″, where each R″ is independently an alkyl. As another example,“haloalkoxy” or “haloalkyloxy” refers to a group of the formula —OR′″,where R′″ is a haloalkyl.

“Protecting group” refers to a group of atoms that, when attached to areactive functional group in a molecule, mask, reduce or prevent thereactivity of the functional group. Typically, a protecting group may beselectively removed as desired during the course of a synthesis.Examples of protecting groups can be found in Greene and Wuts,Protective Groups in Organic Chemistry, 3^(rd) Ed., 1999, John Wiley &Sons, NY and Harrison et al., Compendium of Synthetic Organic Methods,Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Representative nitrogenprotecting groups include, but are not limited to, formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl(“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl(“TES”), trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl(“NVOC”) and the like. Representative hydroxyl protecting groupsinclude, but are not limited to, those where the hydroxyl group iseither acylated (esterified) or alkylated such as benzyl and tritylethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilylethers (e.g., TMS or TIPPS groups), glycol ethers, such as ethyleneglycol and propylene glycol derivatives and allyl ethers.

The present invention provides compounds and pharmaceutical compositionsuseful for the treatment of various disease states and conditions,having the formula:

wherein P₁ and P₂ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein R₁ and R₂ each individually are substituted or unsubstituted,branched or unbranched alkyl groups, substituted or unsubstituted arylgroups, substituted or unsubstituted, branched or unbranched alkylarylgroups, halogen atoms, hydrogen atoms or combinations thereof;

wherein Z is —C(O)OR^(d), —C(O)NR^(c)R^(c), —C(O)H, —C(NH)NR^(c)R^(c),—C(S)H, —C(S)OR^(d), —C(S)NR^(c)R^(c), —CN;

each R^(a), is independently selected from the group consisting ofhydrogen, (C1-C6) alkyl, (C3-C8) cycloalkyl, cyclohexyl, (C4-C11)cycloalkylalkyl, (C5-C10) aryl, phenyl, (C6-C16) arylalkyl, benzyl, 2-6membered heteroalkyl, 3-8 membered cycloheteroalkyl, morpholinyl,piperazinyl, homopiperazinyl, piperidinyl, 4-11 memberedcycloheteroalkylalkyl, 5-10 membered heteroaryl and 6-16 memberedheteroarylalkyl;

each R^(b), is a suitable group independently selected from the groupconsisting of ═O, —OR^(d), (C1-C3) haloalkyloxy, —OCF₃, ═S, —SR^(d),═NR^(d), ═NOR^(d), —NR^(c)R^(c), halogen, —CF₃, —CN, —NC, —OCN, —SCN,—NO, —NO₂, ═N₂, —N₃, —S(O)R^(d), —S(O)₂R^(d), —S(O)₂OR^(d),—S(O)NR^(c)R^(c), —S(O)₂NR^(c)R^(c), —OS(O)R^(d), —OS(O)₂R^(d),—OS(O)₂OR^(d), —OS(O)₂NR^(c)R^(c), —C(O)R^(d), —C(O)OR^(d),—C(O)NR^(c)R^(c), —C(NH)NR^(c)R^(c), —C(NR^(a))NR^(c)R^(c),—C(NOH)R^(a), —C(NOH)NR^(c)R^(c), —OC(O)R^(d), —OC(O)OR^(d),—OC(O)NR^(c)R^(c), —OC(NH)NR^(c)R^(c), —OC(NR^(a))NR^(c)R^(c),—[NHC(O)]_(n)R^(d), —[NR^(a)C(O)]_(n)R^(d), —[NHC(O)]_(n)OR^(d),—[NR^(a)C(O)]_(n)OR^(d), —[NHC(O)]_(n)NR^(c)R^(c),—[NR^(a)C(O)]_(n)NR^(c)R^(c), —[NHC(NH)]_(n)NR^(c)R^(c) and—[NR^(a)C(NR^(a))]_(n)NR^(c)R^(c);

each R^(c), is independently a protecting group or R^(a), or,alternatively, each R^(c) is taken together with the nitrogen atom towhich it is bonded to form a 5 to 8-membered cycloheteroalkyl orheteroaryl which may optionally include one or more of the same ordifferent additional heteroatoms and which may optionally be substitutedwith one or more of the same or different R^(a) or suitable R^(b)groups;

each n, independently is an integer from 0 to 3;

each R^(d), independently is a protecting group or R^(a);

in particular, Z is a carboxylic acid, ester, amide, thiocarbamate,carbamate, thioester, thiocarboxamide or a nitrile;

and pharmaceutically acceptable salts thereof. In certain embodiments,P₁ and P₂ are hydrogen atoms, R₁ and R₂ each individually are methylgroups or hydrogen atoms or combinations thereof, and Z is carboxylicacid or a carboxylic ester.

The present invention also provides compounds and pharmaceuticalcompositions useful for the treatment of various disease states andconditions, having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein R₁ and R₂ each individually are substituted or unsubstituted,branched or unbranched alkyl groups, substituted or unsubstituted arylgroups, substituted or unsubstituted, branched or unbranched alkylarylgroups, halogen atoms, hydrogen atoms or combinations thereof;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In certain embodiments,P₁, P₂ and P₃ each are hydrogen atoms, R₁ and R₂ each individually aremethyl groups or hydrogen atoms or combinations thereof and Z is acarboxylic acid or a carboxylic ester.

In certain aspects the designation of OP₃ serves to denote that theterminal carbon is substituted with one or more halogens (I, Cl, F, Br,mono, di or tri substitution) to form, for example, a trifluoromethylgroup, or is an aryl group or phenoxy group that can be substituted orunsubstituted as described herein.

The present invention still further provides compounds andpharmaceutical compositions useful for the treatment of various diseasestates and conditions, having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein R₁ is a substituted or unsubstituted, branched or unbranchedalkyl group, substituted or unsubstituted aryl group, substituted orunsubstituted, branched or unbranched alkylaryl group, halogen atom, ora hydrogen atom;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In an embodiment, P₁, P₂and P₃ each are hydrogen atoms, R₁ is a methyl group or a hydrogen atomand Z is a carboxylic acid or a carboxylic ester.

The present invention provides compounds and pharmaceutical compositionsuseful for the treatment of various disease states and conditions,having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein R₁ is a substituted or unsubstituted, branched or unbranchedalkyl group, substituted or unsubstituted aryl group, substituted orunsubstituted, branched or unbranched alkylaryl group, halogen atom, ora hydrogen atom;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In a particularembodiment, P₁, P₂ and P₃ each are hydrogen atoms, R₁ is a methyl groupor a hydrogen atom and Z is a carboxylic acid or a carboxylic ester.

The present invention further provides compounds and pharmaceuticalcompositions useful for the treatment of various disease states andconditions, having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein R₁, R₂ and R₃, each individually are substituted orunsubstituted, branched or unbranched alkyl groups, substituted orunsubstituted aryl groups, substituted or unsubstituted, branched orunbranched alkylaryl groups, halogen atoms, hydrogen atoms orcombinations thereof;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In a particularembodiment, P₁, P₂ and P₃ each are hydrogen atoms, R₁, R₂ and R₃ eachindividually are methyl groups or hydrogen atoms or combinations thereofand Z is a carboxylic acid or a carboxylic ester.

The present invention also provides compounds and pharmaceuticalcompositions useful for the treatment of various disease states andconditions, having the formula:

wherein P₁ and P₂ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein R₁ and R₂ each individually are substituted or unsubstituted,branched or unbranched alkyl groups, substituted or unsubstituted arylgroups, substituted or unsubstituted, branched or unbranched alkylarylgroups, halogen atoms, hydrogen atoms or combinations thereof;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In certain embodiments,P₁ and P₂ are hydrogen atoms, R₁ and R₂ each individually are methylgroups or hydrogen atoms or combinations thereof and Z is carboxylicacid or a carboxylic ester.

The present invention provides compounds and pharmaceutical compositionsuseful for the treatment of various disease states and conditions,having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein R₁ and R₂ each individually are substituted or unsubstituted,branched or unbranched alkyl groups, substituted or unsubstituted arylgroups, substituted or unsubstituted, branched or unbranched alkylarylgroups, halogen atoms, hydrogen atoms or combinations thereof;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In a particularembodiment, P₁, P₂ and P₃ each are hydrogen atoms and Z is a carboxylicacid or a carboxylic ester.

In certain aspects the designation of OP₃ serves to denote that theterminal carbon is substituted with one or more halogens (I, Cl, F, Br,mono, di or tri substitution) to form, for example, a trifluoromethylgroup, or is an aryl group or phenoxy group that can be substituted orunsubstituted as described herein.

The present invention also provides compounds and pharmaceuticalcompositions useful for the treatment of various disease states andconditions, having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein R₁, R₂ and R₃, each individually are substituted orunsubstituted, branched or unbranched alkyl groups, substituted orunsubstituted aryl groups, substituted or unsubstituted, branched orunbranched alkylaryl groups, halogen atoms, hydrogen atoms orcombinations thereof;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one embodiment, P₁, P₂and P₃ each are hydrogen atoms, R₁, R₂ and R₃ each individually aremethyl groups or hydrogen atoms or combinations thereof and Z is acarboxylic acid or a carboxylic ester.

The present invention further still provides compounds andpharmaceutical compositions useful for the treatment of various diseasestates and conditions, having the formula:

wherein P₁ and P₂ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein R₁ and R₂ each individually are substituted or unsubstituted,branched or unbranched alkyl groups, substituted or unsubstituted arylgroups, substituted or unsubstituted, branched or unbranched alkylarylgroups, halogen atoms, hydrogen atoms or combinations thereof;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one particularembodiment, P₁ and P₂ are hydrogen atoms, R₁ and R₂ each individuallyare methyl groups or hydrogen atoms or combinations thereof and Z iscarboxylic acid or a carboxylic ester.

The present invention further still provides compounds andpharmaceutical compositions useful for the treatment of various diseasestates and conditions, having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein R₁ is a substituted or unsubstituted, branched or unbranchedalkyl group, substituted or unsubstituted aryl group, substituted orunsubstituted, branched or unbranched alkylaryl group, halogen atom, ora hydrogen atom;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one embodiment, P₁, P₂and P₃ each are hydrogen atoms, R₁ is a methyl group or a hydrogen atomand Z is a carboxylic acid or a carboxylic ester.

The present invention provides compounds and pharmaceutical compositionsuseful for the treatment of various disease states and conditions,having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein R₁ is a substituted or unsubstituted, branched or unbranchedalkyl group, substituted or unsubstituted aryl group, substituted orunsubstituted, branched or unbranched alkylaryl group, halogen atom, ora hydrogen atom;

wherein Q represents one or more substitutents and each Q,independently, is a hydrogen atom, a halogen atom or a branched orunbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl,cycloalkyl, aryl, alkoxy, aryloxy, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, cyano, carboxyl,alkoxycarbonyloxy, aryloxycarbonyloxy or aminocarbonyl group;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one particular aspect,P₁, P₂ and P₃ each are hydrogen atoms, R₁ is a methyl group or ahydrogen atom, each Q is a hydrogen atom and Z is a carboxylic acid or acarboxylic ester.

The present invention also provides compounds and pharmaceuticalcompositions useful for the treatment of various disease states andconditions, having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein R₁ is a substituted or unsubstituted, branched or unbranchedalkyl group, substituted or unsubstituted aryl group, substituted orunsubstituted, branched or unbranched alkylaryl group, halogen atom, ora hydrogen atom;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one embodiment, P₁, P₂and P₃ each are hydrogen atoms, R₁ is a methyl group or a hydrogen atom,and Z is a carboxylic acid or a carboxylic ester.

The present invention further still provides compounds andpharmaceutical compositions useful for the treatment of various diseasestates and conditions, having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein R₁ is a substituted or unsubstituted, branched or unbranchedalkyl group, substituted or unsubstituted aryl group, substituted orunsubstituted, branched or unbranched alkylaryl group, halogen atom, ora hydrogen atom;

wherein Q represents one or more substitutents and each Q,independently, is a hydrogen atom, halogen atom or a branched orunbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl,cycloalkyl, aryl, alkoxy, aryloxy, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, cyano, carboxyl,alkoxycarbonyloxy, aryloxycarbonyloxy or aminocarbonyl group;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one particularembodiment, P₁, P₂ and P₃ each are hydrogen atoms, R is a methyl groupor a hydrogen atom, each Q is a hydrogen atom and Z is a carboxylic acidor a carboxylic ester.

In still another aspect, the present invention provides compounds andpharmaceutical compositions useful for the treatment of various diseasestates and conditions, having the formula:

wherein P₁, and P₂ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein R₁ and R₂ each individually are substituted or unsubstituted,branched or unbranched alkyl groups, substituted or unsubstituted arylgroups, substituted or unsubstituted, branched or unbranched alkylarylgroups, halogen atoms, hydrogen atoms or combinations thereof;

wherein Q represents one or more substitutents and each Q,independently, is a hydrogen atom, halogen atom or a branched orunbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl,cycloalkyl, aryl, alkoxy, aryloxy, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, cyano, carboxyl,alkoxycarbonyloxy, aryloxycarbonyloxy or aminocarbonyl group;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one embodiment, P₁,and P₂ each are hydrogen atoms, R₁ and R₂ each individually are methylgroups or hydrogen atoms or combinations thereof, each Q is a hydrogenatom and Z is a carboxylic acid or a carboxylic ester.

The present invention also provides compounds and pharmaceuticalcompositions useful for the treatment of various disease states andconditions, having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein R₁, R₂ and R₃, each individually are substituted orunsubstituted, branched or unbranched alkyl groups, substituted orunsubstituted aryl groups, substituted or unsubstituted, branched orunbranched alkylaryl groups, halogen atoms, hydrogen atoms orcombinations thereof;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one embodiment, P₁, P₂and P₃ each are hydrogen atoms, R₁, R₂ and R₃ each individually aremethyl groups or hydrogen atoms or combinations thereof and Z is acarboxylic acid or a carboxylic ester.

The present invention further provides compounds and pharmaceuticalcompositions useful for the treatment of various disease states andconditions, having the formula:

wherein P₁, and P₂ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein R₁ and R₂ each individually are substituted or unsubstituted,branched or unbranched alkyl groups, substituted or unsubstituted arylgroups, substituted or unsubstituted, branched or unbranched alkylarylgroups, halogen atoms, hydrogen atoms or combinations thereof;

wherein U is a branched or unbranched, substituted or unsubstitutedalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkoxy, aryloxy,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl,alkoxycarbonyloxy, and aryloxycarbonyloxy group;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one aspect, P₁, and P₂each are hydrogen atoms, R₁ and R₂ each individually are methyl groupsor hydrogen atoms or combinations thereof, U is a trifluoromethyl groupand Z is a carboxylic acid or a carboxylic ester.

In still another aspect, the present invention provides compounds andpharmaceutical compositions useful for the treatment of various diseasestates and conditions, having the formula:

wherein P₁, and P₂ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein R₁ and R₂ each individually are substituted or unsubstituted,branched or unbranched alkyl groups, substituted or unsubstituted arylgroups, substituted or unsubstituted, branched or unbranched alkylarylgroups, halogen atoms, hydrogen atoms or combinations thereof;

wherein U is a branched or unbranched, substituted or unsubstitutedalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkoxy, aryloxy,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl,alkoxycarbonyloxy, and aryloxycarbonyloxy group;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. For example, P₁, and P₂each are hydrogen atoms, R₁ and R₂ each individually are methyl groupsor hydrogen atoms or combinations thereof, U is a trifluoromethyl groupand Z is a carboxylic acid or a carboxylic ester.

The present invention provides compounds and pharmaceutical compositionsuseful for the treatment of various disease states and conditions,having the formula:

wherein P₁ and P₂ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In certain embodiments,P₁ and P₂ are hydrogen atoms and Z is carboxylic acid or a carboxylicester.

The analogs are designated as 7,17-diHDHAs. In certain aspects, thechiral carbon atom at the 7 position (C-7) has an R configuration. Inanother aspect, the C-7 carbon atom preferably has an S configuration.In still another aspect, the C-7 carbon atom is as an R/S racemate.Additionally, the chiral carbon atom at the 17 position (C-17) can havean R configuration. Alternatively, the C-17 carbon can have an Sconfiguration. In still yet another aspect, the C-17 carbon canpreferably exist as an R/S racemate. Exemplary analogs include, forexample, 7S,17R/S-diHDHA,7S,17R/S-dihydroxy-docosa-4Z,8E,10Z,13Z,15E,19Z-hexaenoic acid.

The present invention also provides compounds and pharmaceuticalcompositions useful for the treatment of various disease states andconditions, having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In certain embodiments,P₁, P₂ and P₃ each are hydrogen atoms and Z is a carboxylic acid or acarboxylic ester.

In certain aspects the designation of OP₃ serves to denote that theterminal carbon is substituted with one or more halogens (I, Cl, F, Br,mono, di or tri substitution) to form, for example, a trifluoromethylgroup, or is an aryl group or phenoxy group that can be substituted orunsubstituted as described herein.

The present invention further provides compounds and pharmaceuticalcompositions useful for the treatment of various disease states andconditions, having the formula:

wherein P₁ is a protecting group or a hydrogen atom;

wherein X is a substituted or unsubstituted methylene, an oxygen atom, asubstituted or unsubstituted nitrogen atom, or a sulfur atom;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one embodiment, P₁ isa hydrogen atom, X is an oxygen atom and Z is a carboxylic acid or acarboxylic ester.

The present invention still further provides compounds andpharmaceutical compositions useful for the treatment of various diseasestates and conditions, having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In an embodiment, P₁, P₂and P₃ each are hydrogen atoms and Z is a carboxylic acid or acarboxylic ester.

The analogs are designated as 7,8,17-trihydroxy-DHAs. In certainembodiments, the chiral carbon atom at the 7 position (C-7) has an Rconfiguration. In other embodiments, the C-7 carbon atom preferably hasan S configuration. In still other embodiments, the C-7 carbon atom isas an R/S racemate. In certain aspects, the chiral carbon atom at the 8position (C-8) has an R configuration. In another aspect, the C-8 carbonatom has an S configuration. In still another aspect, the C-8 carbonatom preferably is as an R/S racemate. Additionally, the chiral carbonatom at the 17 position (C-17) can have an R configuration.Alternatively, the C-17 carbon can preferably have an S configuration.In still yet another aspect, the C-17 carbon can exist as an R/Sracemate.

The present invention provides compounds and pharmaceutical compositionsuseful for the treatment of various disease states and conditions,having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In a particularembodiment, P₁, P₂ and P₃ each are hydrogen atoms and Z is a carboxylicacid or a carboxylic ester.

The analogs are designated as 7,16,17-trihydroxy-DHAs. In certainembodiments, the chiral carbon atom at the 7 position (C-7) has an Rconfiguration. In other embodiments, the C-7 carbon atom preferably hasan S configuration. In still other embodiments, the C-7 carbon atom isas an R/S racemate. In certain aspects, the chiral carbon atom at the 16position (C-16) has an R configuration. In another aspect, the C-16carbon atom has an S configuration. In still another aspect, the C-16carbon atom preferably is as an R/S racemate. Additionally, the chiralcarbon atom at the 17 position (C-17) can have an R configuration.Alternatively, the C-17 carbon can preferably have an S configuration.In still yet another aspect, the C-17 carbon can exist as an R/Sracemate.

The present invention also provides compounds and pharmaceuticalcompositions useful for the treatment of various disease states andconditions, having the formula:

wherein P₁ is a protecting group or a hydrogen atom;

wherein X is a substituted or unsubstituted methylene, an oxygen atom, asubstituted or unsubstituted nitrogen atom, or a sulfur atom;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one embodiment, P₁ isa hydrogen atom, X is an oxygen atom and Z is a carboxylic acid or acarboxylic ester.

The present invention further provides compounds and pharmaceuticalcompositions useful for the treatment of various disease states andconditions, having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In a particularembodiment, P₁, P₂ and P₃ each are hydrogen atoms and Z is a carboxylicacid or a carboxylic ester.

The analogs are designated as 4,11,17-trihydroxy-DHAs. In certainembodiments, the chiral carbon atom at the 4 position (C-4) has an Rconfiguration. In other embodiments, the C-4 carbon atom preferably hasan S configuration. In still other embodiments, the C-4 carbon atom isas an R/S racemate. In certain aspects, the chiral carbon atom at the 11position (C-11) has an R configuration. In another aspect, the C-11carbon atom has an S configuration. In still another aspect, the C-11carbon atom preferably is as an R/S racemate. Additionally, the chiralcarbon atom at the 17 position (C-17) can have an R configuration.Alternatively, the C-17 carbon can preferably have an S configuration.In still yet another aspect, the C-17 carbon can exist as an R/Sracemate.

The present invention also provides compounds and pharmaceuticalcompositions useful for the treatment of various disease states andconditions, having the formula:

wherein P₁ and P₂ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In certain embodiments,P₁ and P₂ are hydrogen atoms and Z is carboxylic acid or a carboxylicester.

The present invention provides compounds and pharmaceutical compositionsuseful for the treatment of various disease states and conditions,having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In a particularembodiment, P₁, P₂ and P₃ each are hydrogen atoms and Z is a carboxylicacid or a carboxylic ester.

In certain aspects the designation of OP₃ serves to denote that theterminal carbon is substituted with one or more halogens (I, Cl, F, Br,mono, di or tri substitution) to form, for example, a trifluoromethylgroup, or is an aryl group or phenoxy group that can be substituted orunsubstituted as described herein.

The present invention provides compounds and pharmaceutical compositionsuseful for the treatment of various disease states and conditions,having the formula:

wherein P₁ is a protecting group or a hydrogen atom;

wherein X is a substituted or unsubstituted methylene, an oxygen atom, asubstituted or unsubstituted nitrogen atom, or a sulfur atom;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one embodiment P₁ is ahydrogen atom, X is an oxygen atom and Z is a carboxylic acid or acarboxylic ester.

The present invention also provides compounds and pharmaceuticalcompositions useful for the treatment of various disease states andconditions, having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one embodiment, P₁, P₂and P₃ each are hydrogen atoms and Z is a carboxylic acid or acarboxylic ester.

The present invention further still provides compounds andpharmaceutical compositions useful for the treatment of various diseasestates and conditions, having the formula:

wherein P₁ and P₂ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one particularembodiment, P₁ and P₂ are hydrogen atoms and Z is carboxylic acid or acarboxylic ester.

In one particular embodiment, the compound is 5S, 18 (+/−)-diHEPA,wherein the carboxyl group can be an acid, ester or salt. 5S, 18(+/−)-diHEPA has been synthesized with 5-lipoxygenase potato and itsphysical properties are depicted in FIG. 15 based on LC-MS-MS.Additionally, 5S, 18 (+/−)-diHEPA is biologically active and hasanti-inflammatory activity as noted by the downregulation of PMNinfiltration in a peritonitis model. It is equipotent to Resolvin E1 atan equal dose amount (See FIG. 16).

The present invention further still provides compounds andpharmaceutical compositions useful for the treatment of various diseasestates and conditions, having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one embodiment, P₁, P₂and P₃ each are hydrogen atoms and Z is a carboxylic acid or acarboxylic ester.

The present invention provides compounds and pharmaceutical compositionsuseful for the treatment of various disease states and conditions,having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein Q independently is a hydrogen atom, halogen atom or a branchedor unbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl,cycloalkyl, aryl, alkoxy, aryloxy, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, cyano, carboxyl,alkoxycarbonyloxy, aryloxycarbonyloxy or aminocarbonyl group;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one particular aspect,P₁, P₂ and P₃ each are hydrogen atoms, each Q is a hydrogen atom and Zis a carboxylic acid or a carboxylic ester.

The present invention also provides compounds and pharmaceuticalcompositions useful for the treatment of various disease states andconditions, having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one embodiment, P₁, P₂and P₃ each are hydrogen atoms and Z is a carboxylic acid or acarboxylic ester.

The present invention further still provides compounds andpharmaceutical compositions useful for the treatment of various diseasestates and conditions, having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein Q represents one or more substitutents and each Q,independently, is a hydrogen atom, halogen atom or a branched orunbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl,cycloalkyl, aryl, alkoxy, aryloxy, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, cyano, carboxyl,alkoxycarbonyloxy, aryloxycarbonyloxy or aminocarbonyl group;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one particularembodiment, P₁, P₂ and P₃ each are hydrogen atoms, each Q is a hydrogenatom and Z is a carboxylic acid or a carboxylic ester.

In still another aspect, the present invention provides compounds andpharmaceutical compositions useful for the treatment of various diseasestates and conditions, having the formula:

wherein P₁, and P₂ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein Q represents one or more substitutents and each Q,independently, is a hydrogen atom, halogen atom or a branched orunbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl,cycloalkyl, aryl, alkoxy, aryloxy, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, cyano, carboxyl,alkoxycarbonyloxy, aryloxycarbonyloxy or aminocarbonyl group;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one embodiment, P₁,and P₂ each are hydrogen atoms, each Q is a hydrogen atom and Z is acarboxylic acid or a carboxylic ester.

In still another aspect, the present invention provides compounds andpharmaceutical compositions useful for the treatment of various diseasestates and conditions, having the formula:

wherein P₁ is a protecting group or a hydrogen atom;

wherein X is a substituted or unsubstituted methylene, an oxygen atom, asubstituted or unsubstituted nitrogen atom, or a sulfur atom;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one aspect, P₁ is ahydrogen atom, X is an oxygen atom and Z is a carboxylic acid or acarboxylic ester.

The present invention also provides compounds and pharmaceuticalcompositions useful for the treatment of various disease states andconditions, having the formula:

wherein P₁, P₂ and P₃ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one embodiment, P₁, P₂and P₃ each are hydrogen atoms and Z is a carboxylic acid or acarboxylic ester.

The present invention further provides compounds and pharmaceuticalcompositions useful for the treatment of various disease states andconditions, having the formula:

wherein P₁, and P₂ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein U is a branched or unbranched, substituted or unsubstitutedalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkoxy, aryloxy,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl,alkoxycarbonyloxy, and aryloxycarbonyloxy group;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. In one aspect, P₁, and P₂each are hydrogen atoms, U is a trifluoromethyl group and Z is acarboxylic acid or a carboxylic ester.

In still another aspect, the present invention provides compounds andpharmaceutical compositions useful for the treatment of various diseasestates and conditions, having the formula:

wherein P₁, and P₂ each individually are protecting groups, hydrogenatoms or combinations thereof;

wherein U is a branched or unbranched, substituted or unsubstitutedalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkoxy, aryloxy,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl,alkoxycarbonyloxy, and aryloxycarbonyloxy group;

wherein Z is as defined above, and in particular can be a carboxylicacid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamideor a nitrile;

and pharmaceutically acceptable salts thereof. For example, P₁, and P₂each are hydrogen atoms, U is a trifluoromethyl group and Z is acarboxylic acid or a carboxylic ester.

“Q” includes, for example, hydrogen atoms, halogens (fluorine, iodine,chlorine, bromine), alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkoxy,aryloxy, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl,amino, hydroxy, cyano, carboxyl, alkoxycarbonyloxy, aryloxycarbonyloxyor aminocarbonyl. The organic moieties described herein can further bebranched or unbranched, substituted or unsubstituted.

“U” includes, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,alkoxy, aryloxy, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,aryloxycarbonyl, alkoxycarbonyloxy, and aryloxycarbonyloxy. The organicmoieties described herein can further be branched or unbranched,substituted or unsubstituted.

In one embodiment, the present invention pertains to monohydroxydocosahexaenoic acid (DHA) analogs having the formula

The analogs are designated as 4-hydroxy-DHAs, where P₁ is a protectinggroup or a hydrogen atom and Z is as defined above. In particular, Z canbe a carboxylic acid, ester, amide, thiocarbamate, carbamate, thioester,thiocarboxamide or a nitrile. In one embodiment, the chiral carbon atomat the 4 position (C-4) has an S configuration. In another embodiment,the C-4 carbon atom has an R configuration. In still another embodiment,the C-4 carbon atom is as a racemic mixture, e.g., an R/S configuration.In particular, the present invention includes monohydroxy DHA analogs(as described herein) of 4S-HDHA, 4S-hydroxy-docosa-5E,7Z,10Z,13Z,16Z,19Z-hexaenoic acid and salts and esters thereof.

In another embodiment, the monohydroxy DHA is7S-monohydroxy-docosahexaenoic acid. The analogs are designated as7-hydroxy-DHAs, where P₁ (where the hydroxyl is protected) and Z are asdefined above. In particular, Z can be a carboxylic acid, ester, amide,thiocarbamate, carbamate, thioester, thiocarboxamide or a nitrile. Inone embodiment, the chiral carbon atom at the 7 position (C-7) has an Sconfiguration. In another embodiment, the C-7 carbon atom has an Rconfiguration. In still another embodiment, the C-7 carbon atom is as aracemic mixture, e.g., an R/S configuration.

The present invention, further pertains to dihydroxy-docosahexaenoicacid analogs (diHDHA) having the formula

The analogs are designated as 10,17-diHDHAs. P₁ and P₂ are as definedabove and can be the same or different. Z is as defined above and inparticular can be a carboxylic acid, ester, amide, thiocarbamate,carbamate, thioester, thiocarboxamide or a nitrile. The broken doublebond line indicates that either the E or Z isomer is within the scope ofthe analog(s). In certain aspects, the chiral carbon atom at the 10position (C-10) has an R configuration. In another aspect, the C-10carbon atom has an S configuration. In still another aspect, the C-10carbon atom preferably is as an R/S racemate. Additionally, the chiralcarbon atom at the 17 position (C-17) can have an R configuration.Alternatively, the C-17 carbon can preferably have an S configuration.In still yet another aspect, the C-17 carbon can exist as an R/Sracemate. In one example, the present invention includes10,17S-docosatriene,10,17S-dihydroxy-docosa-4Z,7Z,11E,13,15E,19Z-hexaenoic acid analogs suchas 10R/S-OCH₃,17S-HDHA, 10R/S, methoxy-17Shydroxy-docosa-4Z,7Z,11E,13,15E,19Z-hexaenoic acid derivatives.

In still yet another embodiment, the present invention pertains todiHDHA analogs having the formula

The analogs are designated as 4,17-diHDHAs. P₁, P₂ and Z are as definedabove. P₁ and P₂ can be the same or different. In particular, Z can be acarboxylic acid, ester, amide, thiocarbamate, carbamate, thioester,thiocarboxamide or a nitrile. In certain aspects, the chiral carbon atomat the 4 position (C-4) has an R configuration. In another aspect, theC-4 carbon atom preferably has an S configuration. In still anotheraspect, the C-4 carbon atom is as an R/S racemate. Additionally, thechiral carbon atom at the 17 position (C-17) can have an Rconfiguration. Alternatively, the C-17 carbon can have an Sconfiguration. In still yet another aspect, the C-17 carbon canpreferably exist as an R/S racemate.

For example, the present invention includes 4S, 17R/S— diHDHA,4S,17R/S-dihydroxy-docosa-5E,7Z,10Z,13Z,15E,19Z-hexaenoic acid analogs.

It should be understood that “Z” can be altered from one particularmoiety to another by a skilled artisan. In order to accomplish this insome particular instances, one or more groups may require protection.This is also within the skill of an ordinary artisan. For example, acarboxylic ester (Z) can be converted to an amide by treatment with anamine. Such interconversion are known in the art.

In the EPA and DHA analogs, it should be understood that reference to“hydroxyl” stereochemistry is exemplary, and that the term is meant toinclude protected hydroxyl groups as well as the free hydroxyl group. Incertain embodiments, the C-17 position has an R configuration. In otherembodiment, the C-17 position has an S configuration. In other aspects,certain embodiments of the invention have an R configuration at the C-18position.

In certain aspects of the present invention, ASA pathways generate R>Sand therefore, 4S, 5R/S, 7S,8R/S, 11R,12R/S 16 S,17R. With respect tospecies generated from the 15-LO pathway the chirality of C-17 is S,C-16R and C-10, preferably R.

In certain embodiments, the invention does not include5,12,18-trihydroxy-EPA, and more specifically, does not include6,8,10,14,16-eicosapentaenoic acid, 5,12,18-trihydroxy and analogsthereof, as described in U.S. patent application Ser. No. 09/785,866,filed Feb. 16, 2001.

The hydroxyl(s) in the EPA and DHA analogs can be protected by variousprotecting groups (P), such as those known in the art. An artisanskilled in the art can readily determine which protecting group(s) maybe useful for the protection of the hydroxyl group(s). Standard methodsare known in the art and are more fully described in literature. Forexample, suitable protecting groups can be selected by the skilledartisan and are described in Green and Wuts, “Protecting Groups inOrganic Synthesis”, John Wiley and Sons, Chapters 5 and 7, 1991, theteachings of which are incorporated herein by reference. Preferredprotecting groups include methyl and ethyl ethers, TMS or TIPPS groups,acetate (esters) or proprionate groups and glycol ethers, such asethylene glycol and propylene glycol derivatives.

For example, one or more hydroxyl groups can be treated with a mildbase, such as triethylamine in the presence of an acid chloride or silylchloride to facilitate a reaction between the hydroxyl ion and thehalide. Alternatively, an alkyl halide can be reacted with the hydroxylion (generated by a base such as lithium diisopropyl amide) tofacilitate ether formation.

It should also be understood that for the EPA and DHA analogs, not allhydroxyl groups need be protected. One, two or all three hydroxyl groupscan be protected. This can be accomplished by the stoichiometric choiceof reagents used to protect the hydroxyl groups. Methods known in theart can be used to separate the mono, di- or tri-protected hydroxycompounds, e.g., HPLC, LC, flash chromatography, gel permeationchromatography, crystallization, distillation, etc.

It should be understood that there are one or more chiral centers ineach of the above-identified compounds. It should understood that thepresent invention encompasses all stereochemical forms, e.g.,enantiomers, diastereomers and racemates of each compound. Whereasymmetric carbon atoms are present, more than one stereoisomer ispossible, and all possible isomeric forms are intended to be includedwithin the structural representations shown. Optically active (R) and(S) isomers may be resolved using conventional techniques known to theordinarily skilled artisan. The present invention is intended to includethe possible diastereiomers as well as the racemic and opticallyresolved isomers.

The resolvin analogs depicted throughout the specification containacetylenic and/or ethylenically unsaturated sites. Where carbon carbondouble bonds exist, the configurational chemistry can be either cis (Z)or trans (E) and the depictions throughout the specification are notmeant to be limiting. The depictions are, in general, presented basedupon the configurational chemistry of related DHA or EPA compounds, andalthough not to be limited by theory, are believed to possess similarconfiguration chemistry.

Throughout the specification carbon carbon bonds in particular have been“distorted” for ease to show how the bonds may ultimately be positionedrelative one to another. For example, it should be understood thatacetylenic portions of the resolvins actually do include a geometry ofapproximately 180 degress, however, for aid in understanding of thesynthesis and relationship between the final product(s) and startingmaterials, such angles have been obfuscated to aid in comprehension.

Throughout the organic synthesis presented below, it should beunderstood that hydrogenation of acetylenic portions of the resolvinanalog may result in one or more products.

It is intended that all possible products are included within thisspecification. For example, hydrogenation of a diacetylenic resolvinanalog can produce up to 8 products (four diene products, i.e., cis,cis; cis, trans; trans, cis; trans, trans) if hydrogenation of bothacetylenic portions is completed (this can be monitored by knownmethods) and four monoacetylene-monoethylene products (cis or trans“monoene”-acetylene; acetylene-cis or trans “monoene”. All products canbe separated and identified by HPLC, GC, MS, NMR, IR.

Known techniques in the art can be used to convert the carboxylicacid/ester functionality of the resolvin analog into carboxamides,thioesters, nitrile, carbamates, thiocarbamates, etc. and areincorporated herein. The appropriate moieties, such as amides, can befurther substituted as is known in the art.

In general, the resolvin analogs of the invention are bioactive asalcohols. Enzymatic action or reactive oxygen species attack at the siteof inflammation or degradative metabolism. Such interactions with thehydroxyl(s) of the resolvin molecule can eventually reduce physiologicalactivity as depicted below:

The use of “R” groups with secondary bioactive alcohols, in particular,serves to increase the bioavailability and bioactivity of the resolvinanalog by inhibiting or diminishing the potential for oxidation of thealcohol to a ketone producing an inactive metabolite. The R “protectinggroups” include, for example, linear and branched, substituted andunsubstituted alkyl groups, aryl groups, alkylaryl groups, phenoxygroups, and halogens.

Generally the use of “R protection chemistry” is not necessary withvicinal diols within the resolvin analog. Typically vicinal diols arenot as easily oxidized and therefore, generally do not require suchprotection by substitution of the hydrogen atom adjacent to the oxygenatom of the hydroxyl group. Although it is generally considered thatsuch protection is not necessary, it is possible to prepare suchcompounds where each of the vicinal diol hydroxyl groups, independently,could be “protected” by the substitution of the hydrogen atom adjacentto the oxygen atom of the hydroxyl group with an “R protecting group” asdescribed above.

The term “tissue” is intended to include intact cells, blood, bloodpreparations such as plasma and serum, bones, joints, muscles, smoothmuscles, and organs.

The term “subject” is intended to include living organisms susceptibleto conditions or diseases caused or contributed bacteria, pathogens,disease states or conditions as generally disclosed, but not limited to,throughout this specification. Examples of subjects include humans,dogs, cats, cows, goats, and mice. The term subject is further intendedto include transgenic species.

When the compounds of the present invention are administered aspharmaceuticals, to humans and mammals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient, i.e., at least one EPA orDHA analog, in combination with a pharmaceutically acceptable carrier.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a compound(s) of thepresent invention within or to the subject such that it can perform itsintended function. Typically, such compounds are carried or transportedfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; and other non-toxic compatible substancesemployed in pharmaceutical formulations.

In certain embodiments, the compounds of the present invention maycontain one or more acidic functional groups and, thus, are capable offorming pharmaceutically acceptable salts with pharmaceuticallyacceptable bases. The term “pharmaceutically acceptable salts, esters,amides, and prodrugs” as used herein refers to those carboxylate salts,amino acid addition salts, esters, amides, and prodrugs of the compoundsof the present invention which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of patientswithout undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use of the compounds of the invention. The term “salts”refers to the relatively non-toxic, inorganic and organic acid additionsalts of compounds of the present invention. These salts can be preparedin situ during the final isolation and purification of the compounds orby separately reacting the purified compound in its free base form witha suitable organic or inorganic acid and isolating the salt thus formed.These may include cations based on the alkali and alkaline earth metals,such as sodium, lithium, potassium, calcium, magnesium and the like, aswell as non-toxic ammonium, quaternary ammonium, and amine cationsincluding, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. (See, for example, Berge S. M.,et al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977; 66:1-19 which isincorporated herein by reference).

The term “pharmaceutically acceptable esters” refers to the relativelynon-toxic, esterified products of the compounds of the presentinvention. These esters can be prepared in situ during the finalisolation and purification of the compounds, or by separately reactingthe purified compound in its free acid form or hydroxyl with a suitableesterifying agent. Carboxylic acids can be converted into esters viatreatment with an alcohol in the presence of a catalyst. The term isfurther intended to include lower hydrocarbon groups capable of beingsolvated under physiological conditions, e.g., alkyl esters, methyl,ethyl and propyl esters.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable forintravenous, oral, nasal, topical, transdermal, buccal, sublingual,rectal, vaginal and/or parenteral administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound whichproduces a therapeutic effect. Generally, out of one hundred percent,this amount will range from about 1 percent to about ninety-nine percentof active ingredient, preferably from about 5 percent to about 70percent, most preferably from about 10 percent to about 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol and glycerol monostearate; absorbents, such as kaolin andbentonite clay; lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients. Inone aspect, a solution of a EPA or DHA analog can be administered as eardrops to treat otitis.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the activecompound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention. Suchsolutions are useful for the treatment of conjunctivitis.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Intravenous injection administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systematically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of ordinary skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous andsubcutaneous doses of the compounds of this invention for a patient,when used for the indicated analgesic effects, will range from about0.0001 to about 100 mg per kilogram of body weight per day, morepreferably from about 0.01 to about 50 mg per kg per day, and still morepreferably from about 0.1 to about 40 mg per kg per day. For example,between about 0.01 microgram and 20 micrograms, between about 20micrograms and 100 micrograms and between about 10 micrograms and 200micrograms of the compounds of the invention are administered per 20grams of subject weight.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

The pharmaceutical compositions of the invention include a“therapeutically effective amount” or a “prophylactically effectiveamount” of one or more of the EPA or DHA analogs of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result, e.g., a diminishment or prevention of effectsassociated with various disease states or conditions. A therapeuticallyeffective amount of the EPA or DHA analog may vary according to factorssuch as the disease state, age, sex, and weight of the individual, andthe ability of the therapeutic compound to elicit a desired response inthe individual. A therapeutically effective amount is also one in whichany toxic or detrimental effects of the therapeutic agent are outweighedby the therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the EPA or DHA analog and the particular therapeuticor prophylactic effect to be achieved, and (b) the limitations inherentin the art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a EPA or DHA analog of theinvention is 0.1-20 mg/kg, more preferably 1-10 mg/kg. It is to be notedthat dosage values may vary with the type and severity of the conditionto be alleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

Delivery of the EPA or DHA analogs of the present invention to the lungby way of inhalation is an important method of treating a variety ofrespiratory conditions (airway inflammation) noted throughout thespecification, including such common local conditions as bronchialasthma and chronic obstructive pulmonary disease. The EPA or DHA analogscan be administered to the lung in the form of an aerosol of particlesof respirable size (less than about 10 μm in diameter). The aerosolformulation can be presented as a liquid or a dry powder. In order toassure proper particle size in a liquid aerosol, as a suspension,particles can be prepared in respirable size and then incorporated intothe suspension formulation containing a propellant. Alternatively,formulations can be prepared in solution form in order to avoid theconcern for proper particle size in the formulation. Solutionformulations should be dispensed in a manner that produces particles ordroplets of respirable size.

Once prepared an aerosol formulation is filled into an aerosol canisterequipped with a metered dose valve. The formulation is dispensed via anactuator adapted to direct the dose from the valve to the subject.

Formulations of the invention can be prepared by combining (i) at leastone EPA or DHA analog in an amount sufficient to provide a plurality oftherapeutically effective doses; (ii) the water addition in an amounteffective to stabilize each of the formulations; (iii) the propellant inan amount sufficient to propel a plurality of doses from an aerosolcanister; and (iv) any further optional components e.g. ethanol as acosolvent; and dispersing the components. The components can bedispersed using a conventional mixer or homogenizer, by shaking, or byultrasonic energy. Bulk formulation can be transferred to smallerindividual aerosol vials by using valve to valve transfer methods,pressure filling or by using conventional cold-fill methods. It is notrequired that a stabilizer used in a suspension aerosol formulation besoluble in the propellant. Those that are not sufficiently soluble canbe coated onto the drug particles in an appropriate amount and thecoated particles can then be incorporated in a formulation as describedabove.

Aerosol canisters equipped with conventional valves, preferably metereddose valves, can be used to deliver the formulations of the invention.Conventional neoprene and buna valve rubbers used in metered dose valvesfor delivering conventional CFC formulations can be used withformulations containing HFC-134a or HFC-227. Other suitable materialsinclude nitrile rubber such as DB-218 (American Gasket and Rubber,Schiller Park, Ill.) or an EPDM rubber such as Vistalon™ (Exxon),Royalene™ (UniRoyal), bunaEP (Bayer). Also suitable are diaphragmsfashioned by extrusion, injection molding or compression molding from athermoplastic elastomeric material such as FLEXOMER™ GERS 1085 NTpolyolefin (Union Carbide).

Formulations of the invention can be contained in conventional aerosolcanisters, coated or uncoated, anodized or unanodized, e.g., those ofaluminum, glass, stainless steel, polyethylene terephthalate.

The formulation(s) of the invention can be delivered to the respiratorytract and/or lung by oral inhalation in order to effect bronchodilationor in order to treat a condition susceptible of treatment by inhalation,e.g., asthma, chronic obstructive pulmonary disease, etc. as describedthroughout the specification.

The formulations of the invention can also be delivered by nasalinhalation as known in the art in order to treat or prevent therespiratory conditions mentioned throughout the specification.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical composition.

The invention features an article of manufacture that contains packagingmaterial and a EPA or DHA analog formulation contained within thepackaging material. This formulation contains an at least one EPA or DHAanalog and the packaging material contains a label or package insertindicating that the formulation can be administered to the subject totreat one or more conditions as described herein, in an amount, at afrequency, and for a duration effective to treat or prevent suchcondition(s). Such conditions are mentioned throughout the specificationand are incorporated herein by reference. Suitable EPA analogs and DHAanalogs are described herein.

More specifically, the invention features an article of manufacture thatcontains packaging material and at least one EPA or DHA analog containedwithin the packaging material. The packaging material contains a labelor package insert indicating that the formulation can be administered tothe subject to asthma in an amount, at a frequency, and for a durationeffective treat or prevent symptoms associated with such disease statesor conditions discussed throughout this specification.

Materials and Methods

Zymosan A, hematin, NADPH, 15-lipoxygenase, ASA and other NSAIDs werefrom Sigma. Potato 5-lipoxygenase (LO), DHA, and EPA were from CaymanChemical Co. (Ann Arbor, Mich.); and other synthetic standards, hydroxyfatty acids and intermediates used for MS identification and fragmention references were purchased from Cascade Biochem Ltd. (Reading, U.K.).Authentic standards for 4S-hydroxy-5E,7Z,10Z,13Z,16Z,19Z-docosahexaenoicacid (4S-HDHA), 17S-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid(17S-HDHA), and the racemate17R/S-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid (denoted17R/S-HDHA) were from Penn Bio-Organics (Bellefonte, Pa.), and NMRanalyses established the respective double bond configurations.Additional materials used in LC-MS-MS analyses were from vendorsreported in Refs. (2, 33).

Incubations

Human PMN were freshly isolated from venous blood of healthy volunteers(that declined taking medication for ˜2 weeks before donation; BWHprotocol #88-02642) by Ficoll gradient and enumerated. Cells weredivided into 50×10⁶ cells, 1 ml DPBS with Ca²⁺ and Mg²⁺, denoted +/+,and incubations (40 min, 37° C.) were carried out with either 17R/S-HDHA(Penn Bio-Organics) or 17R-HDHA with zymosan A (100 ng/ml, Sigma). Humanumbilical vein (HUVEC) or microvascular (HMVEC; Cascade Biologics,Portland. OR) endothelial cells were cultured for transendothelialmigration (34) and incubations with HMVEC monolayers (1, 2, or 3passages) seeded (˜2×10⁵ cells/cm²) on polycarbonate permeable supportspre-coated with 0.1% gelatin for ASA and DHA. For hypoxia experiments,plates of HUVEC treated with TNFα and IL-1β (both 1 ng/ml) were placedin a hypoxic chamber (3 h, 37° C.) and returned to normoxia (21 h, 37°C.). Next, ASA (500 μM, 30 min) was added followed by DHA (−5 μM) andA23187 (2 μM; 60 min, 37° C.). Individual whole brains were rapidlyisolated from sacrificed mice immediately prior to incubations (eachhalf/2 ml), washed with cold DPBS^(+/+) and incubated with ASA (45 min,37° C., 500 μM). Incubations were stopped with 5 ml cold methanol, andheld at −20° C. for analyses.

Recombinant Cyclooxygenase-2 (COX-2) and Product Analyses

Human recombinant COX-2 was overexpressed in Sf9 insect cells (ATCC).The microsomal fractions (˜8 were suspended in Tris (100 mM, pH 8.0) asin (35). ASA was incubated (˜2 mM, 37° C., 30 min) with COX-2 beforeaddition of DHA (10 μM), or in some experiments [1-¹⁴C]-labeled DHA(from American Radiolabeled Chemicals, Inc.), and conversions weremonitored in parallel using [1-¹⁴C]-labeled C20:4 (with ASA) (as in FIG.2; also see text).

Incubations were extracted with deuterium-labeled internal standards(15-HETE and C20:4) for LC-MS-MS analysis as in (33) using a FinniganLCQ liquid chromatography ion trap tandem mass spectrometer (San Jose,Calif.) equipped with a LUNA C18-2 (150×2 mm×5 μm or 100×2 mm×5 μm)column and a rapid spectra scanning UV diode array detector thatmonitored UV absorbance ˜0.2 min prior to samples entering the MS-MS.All intact cell incubations and in vivo exudates were stopped with 2 mlcold methanol and kept at −80° C. for >30 minutes. Samples wereextracted using C18 solid phase extraction and analyzed using GC-MS (seeTable 2) (Hewlett-Packard), thin layer chromatography or tandem liquidchromatography-mass spectrometry (LC-MS-MS). Also, a Chiralcel OB-Hcolumn (4.6×250 mm; J. T. Baker) was used to determine R and S alcoholconfigurations of monohydroxy-PUFA using isocratic mobile phase(hexane:isopropanol; 97.5:2.5, vol:vol, with a 0.6 ml/min flow rate).Detailed procedures for isolation, quantitation and structuraldetermination of lipid-derived mediators were recently reported (36) andused here essentially as reported for elucidation of novel products.Biogenic synthesis of the novel docosanoids were carried out usingisolated enzymes, i.e. potato 5-LO, rhCOX-2 or ASA treated rhCOX-2 and15-LO each incubated in tandem sequential reactions with either DHA or17R-HDHA to produce the novel compounds in scale up quantities forisolation and confirmation of physical and biological properties.

PMN Migration, Murine Air Pouch Exudates and Peritonitis

Human PMN transendothelial migration was quantitated by monitoringmyeloperoxidase (MPO) an azurophilic granule marker as in (34, 37).Inflammatory exudates were initiated with intrapouch injection ofrecombinant mouse TNFα (100 ng/pouch; R&D Systems) into dorsal airpouches (10) of 6-8 wk male FVB mice fed laboratory rodent diet 5001(Lab Diet, Purina Mills) containing less than 0.25% arachidonic acid,1.49% EPA and 1.86% DHA followed by ASA (500 μg) at 3.5 h and 300 μgDHA/pouch at 4 h post-TNFα injection. At 6 h (within the resolutionphase), pouches were lavaged (3 ml saline) and exudate cells wereenumerated. Inhibition of TNFα stimulated (100 ng/pouch) PMNinfiltration with i.v. tail injection of either 17R-HDHA (as preparedwith COX-2 vide infra), 5S,12,18R-HEPE, or a 15-epi-LXA₄ analog weredetermined with pouch lavages taken at 4 h. Peritonitis was performedusing 6 to 8-week-old FVB male mice (Charles River Laboratories) fedlaboratory Rodent Diet 5001 (Purina Mills) that were anesthetized withisoflurane, and compounds to be tested (125 μl) were administeredintravenously. Zymosan A in 1 ml (1 mg/ml) was injected ˜1-1.5 min laterin the peritoneum. Each test compound (100 ng/incubation, i.e. 17R-HDHAin ethanol) or vehicle alone was suspended in ˜5 μl and mixed in sterilesaline 120 μl. Two hours after the intraperitoneal injections, and inaccordance with the Harvard Medical Area Standing Committee on Animalsprotocol #02570, mice were euthanized and peritoneal lavages rapidlycollected for enumeration.

Cell Culture

Human glioma cells DBTRG-05MG cells (ATCC) were cultured as recommendedby ATCC. For analyses, 10×10⁶ cells per well in 6-well plates (Falcon)were stimulated for 16 h with 50 ng/ml of human recombinant TNFα (Gibco)in the presence of specified concentrations of test compounds (ie.,17R-HDHA) or vehicle (0.04% ethanol). Cells were washed in DPBS^(+/+)and harvested in 1 ml of Trizol (Gibco). For RT-PCR, RNA purificationand RT-PCR were performed as in (38). Primers used in amplificationswere: ^(5′)GGAAGATGCTGGTTCCCTGC^(3′) (SEQ ID NO: 1) and^(5′)CAACACGCAGGACAGGTACA^(3′) (SEQ ID NO: 2) for IL-1β;^(5′)TCCACCACCGTGTTGCTGTAG^(3′) (SEQ ID NO: 3); and^(5′)GACCACAGTCCATGACATCACT^(3′) (SEQ ID NO: 4) for GAPDH. PCR productsobtained with these primers were confirmed by sequencing. Analyses wereperformed for both genes (i.e., GAPDH and IL-1β) in the linear range ofthe reaction. Results were analyzed using the NIH Image program(http://rsb.info.nih.govinih-image).

Biogenic Synthesis of Natural Resolvins and Analogs Biogenic Synthesisof 17R-Containing HDHA Products:

For scale-up production of diHDHA products, human recombinantcyclooxygenase-2 (COX-2) was expressed in insect Sf9 cells and isolatedmicrosomal fractions were prepared and suspended in Tris Buffer (100 mM,pH 8.0). Aspirin was added (2 mM) (30 min, RT) to assess 17R HDHAformation from DHA (10 uM) before large scale preparations that wasconfirmed as by LC-MS-MS analysis as in FIG. 2 (see specification).Next, DHA (100 mg from Sigma D-2534) was suspended in EtOH and added at1% v/v to Borate buffer (0.1 mM H₃BO₃, pH 8.5) and vortexed in around-bottom flask (5 to 10 min under a stream of nitrogen) to formmicelle suspensions (optical density >650 nm) to react first with theacetylated-COX-2 (60 min, RT) to generate the 17R oxygenation (seeillustration scheme A).

These reaction mixtures were immediately transferred and spun through aMillipore YM-30 centrifuge column for 20 min, RT. Next, isolated potato5-lipoxygenase purchased from Cayman Chemical was added at 400 ul (inaccordance with each preparation's specific enzymatic activity) to 10 mlreactions for 30 min, 4° C. in a round-bottom flask flushed with O₂rotating in an ice water bath. At time intervals, samples were takenfrom the reactions to monitor product formation using LC-MS-MS withtandem UV spectra recorded online with a PDA detector with a MeOH:H2Omobile phase and linear gradient.

Next, the 4 position and 7 position hydroperoxy adducts respectivelyintroduced into the 17RHDHA substrate by the actions of the5-lipoxygenase were reduced as a mixture with the addition of solidgrains of sodium borohydride (5 min, RT) and the incubations werestopped with the addition of 2 vol cold MeOH. The diHDHA products wereextracted using liquid-liquid acidic ether (pH 3.5) and washed toapproximately neutral pH with water. The structures of the 4S,17R and7S,17R-diHDHA positional isomers were established by LC-MS-MS (usingconditions cited in the specification). These compounds were wellresolved in rp-HPLC using MeOH:H2O (65:35 v/v) for isolation andbiologic actions.

Biogenic Synthesis of 17 S Containing HDHA Products:

The preparation of the 17S products was carried out using sequential15-lipoxyenase (soybean lipoxygenase; Sigma) followed by addition ofpotato 5-lipoxygenase (Cayman Chemical) for scale-up reactions toproduce the 4S, 17S-diHDHA and 7S, 17S-diHDHA shown in scheme B. Both ofthese lipoxygenases insert molecular oxygen predominantly in the Sconfiguration with antarafacial abstraction of hydrogen at specificpositions in DHA (see specification). For these preparations, DHA (100mg) was suspended in 10 ml Borate buffer (0.1M, pH 9.2), vortexed in around bottom flask (250 ml vol) to form micelles, and the soybean15-lipoxygenase was added to the micelle suspension at 4° C. in an icewater bath using spinning rotation for continuous mixing for 30-40 minsto convert DHA to the 17S-H(p) DHA. This hydroperoxy DHA was reducedwith the addition of a few grains of NaBH₄ to the flask to produce thecorresponding 17S hydroxy-DHA (see Scheme B). Next, the isolated potato5-lipoxygenase was added to the flask kept at 4° C., pH 9.0 withrotation and oxygen to insert the 4S hydroperoxy- and 7S hydroperoxy-into 17S-HDHA followed by reduction with NaBH₄. The reactions weremonitored using LC-MSM-MS (vide supra), stopped with 2 vol MeOH, andacidic ether extracted and the positional isomers isolated usingRP-HPLC. These 17S-containing products gave similar biologic in murineinflammation and physical properties (i.e. UV chromophores) to theircorresponding 17R products (see Table 2), but displayed differentretention times in GC-MS.

Biogenic Synthesis of 5S, 18R/S diHEPA and 5,12,18-tri-EPE

(+/−) 18-HEPE was purchased from Cayman Chemical (cat. Number 32840 CASregistry No. 141110-17-0) and used to produce the new EPA-derivedcompounds (see scheme C) by reactions and procedures described (videsupra) using the potato 5-lipoxygenase for scale up reactions. Theracemate 18+/−HEPE (100 ug aliquots in EtOH) was suspended in Boratebuffer (pH 9.2) in round-bottom flasks (250 ml) in 0.1% EtOH v/v,vortexed 5-10 min to form micelles and placed at 4° C., rotating asnoted above in an ice water bath. The 5-lipoxygenase was added in 25 ulaliquots in two consecutive bolus additions for the isolated enzyme. Thefirst bolus initiated reactions leading to production of 5S,18R/S-diHEPE after NaBH₄ reduction as the main product (see scheme C)generated after 30-40 min reactions as monitored by LC-MS-MS. The secondbolus addition of 5-lipoxygenase to the mixture gave rise to the5,12,18R/S-triHEPA via production of a 5(6)-epoxide intermediate formedby the LTA synthase reaction of the potato 5-lipoxygenase at this pH andsubstrate concentration. The epoxide opens in an SN2 type reaction inthe presence of water adding at the least hindered carbon of thecarbonium cation generated at the end (carbon 12) of the conjugatedtriene system (see below, scheme C). The structures were confirmed byLC-MS-MS and isolated by HPLC for assessment of biologic actions.

Examples of Analogs via Biogenic Synthesis Synthesis of4,5-dehydro-7S,17 S-diHDHA

4,5-dehydro Docosahexaenoic Acid (cat number 90312) was purchased fromCayman Chemical (MI) and used without additional purification to produceanalogs for scale-up biological analysis. The 4,5 dehydro DHA in 100 ugaliquot suspensions was prepared in 0.1M Borate Buffer (pH 9.2) in 25 mlround-bottom flask for vortexing and micelle formation before additionof the 15-lipoxygenase in 25 ul aliquots. After reduction with NaBH₄ ofthe hydroperox- added in the S configuration at position 17, thecorresponding alcohol was next converted with the addition of potato5-lipoxygenase followed by reduction to give the 4,5-dehydro 7S,17S-diHDHA (see scheme D). This scheme can also be used to generate thecorresponding 17R containing analogs by substituting the ASA-treatrecombinant COX-2 in the position 1 enzyme instead of 15-lipoxygenase.

Another example of this route for scale-up is given in Scheme E for thebiogenic synthesis of a novel analog, 4,5-dehydro 10,17S-dihydroxy DHA(See also FIG. 17). In short, after addition of 15-LO that was convertedto the 17S adduct, a second addition of the soybean 15-LO gave theLTA4-like synthase reaction to yield the 16(17) epoxide of the4,5dehydro precursor that underwent hydrolysis to give the 4,5-dehydro10,17S-dihydroxy DHA. This product at a 100 ng dose in murine zymosanA-induced peritonitis gave 40% inhibition of the PMN infiltration,indicating that this Resolvin analog is a potent anti-inflammatory agentin vivo.

FIG. 18 demonstrates that 4,17S-diHDHA is approximately half as potentas 10,17-docosatriene. By increasing the dose given by intravenous bolusto 200 ng, the two compounds are essentially equally effective. Theseresults indicate that the 4,17S-diHDHA, although less potent on an equalquantity basis, is essentially equally effective in both regulating andinhibiting leukocyte infiltration and inflammation in the murineperitonitis model.

For FIG. 18, the 4,17S-diHDHA caused dose-dependent inhibition of PMNleukocyte infiltration. 100 ng of 10,17S-docosatrienes caused potentinhibition. Peritonitis was induced in 6 to 8 week old male FVB mice byperitoneal injection of 1 mg Zymosan A. Compounds 4,17S and10,17S-diHDHA were injected by intravenous bolus injection, 1.5 minutesbefore Zymosan A treatment. Two hours afte induction of peritonitis,rapid peritoneal lavages were collected and cell type enumeration wasperformed.

Organic Syntheses of Resolvin Analogs

The following synthetic routes exemplify methods to prepare the resolvinanalog families of interest. The preparations are not intended to belimiting but serve as another means to prepare such analogs along moretraditional practices and should be considered as complementary to thebiogenic syntheses described above. Isolations methods include, columnchromatography, HPLC, GC, crystallizaton and distillation if necessary.Characterization can be accomplished by UV, MS, MS/MS, GC/MS, NMR, etc.One skilled in the art can appreciate the various methods to prepare,isolate and characterize these novel compounds based upon the teachingsherein.

The general synthetic schemes provided below depict methods to preparethe various “classes” or families of resolvins encompassed by thepresent invention. Throughout the syntheses of these families, R groupsare used to indicate that various groups can be appended to the resolvincarbon chain. Each R group is independent selected, can be the same ordifferent, and it can be envisioned that each R group is not necessarilypresent. In those instances, the attachment site would include ahydrogen atom. As described above, the R group is considered a“protecting R group” and can be an substituted or unsubstituted,branched or unbranched alkyl group, arylalkylgroup, alkylaryl group or ahalogen atom.

Throughout the synthetic schemes, various hydroxyl protecting groups aredepicted. These are not to be considered limiting; these are exemplaryprotecting groups that can be used and were chosen as illustrative.

The moiety designated as “U” as used throughout the synthetic schemes isdescribed throughout the application and is incorporated herein byreference. “U” as used throughout the synthetic schemes herein is meantto include a terminal carbon atom. The terminal group can be a mono, dior tri substituted methyl group, a methylene (substituted orunsubstituted) attached to a phenoxy group (substituted orunsubstituted), a substituted or unsubstituted aryl group, arylalkylgroups, etc.

“Q” is defined throughout the specification is intended to include oneor more substituents positioned about a ring structure. Suitablesubstituents include, hydrogen atoms, halogen atoms, alkyl groups(substituted and unsubstituted, branched and unbranched), alkylarylgroups, arylalkyl groups, esters, hydroxyls, etc.

The moiety designated as “X” as used throughout the synthetic schemes isdescribed throughout the application and is incorporated herein byreference. “X” as used throughout the synthetic schemes is intended toinclude, an oxygen atoms, a methylene, a substituted or unsubstituednitrogen atom or sulfur atom.

As described above, hydrogenation of acetylenic portions of the resolvincan be accomplished to provide one or more products. Selectivehydrogenation can provide multiple reaction product dependent upon thedegree of hydrogenation that is desired. The resultant product(s) canprovide one or more geometric isomers (cis or trans) about the resultantdouble bond where hydrogenation has taken place. Additionally, selectivehydrogenation can provide resolvin analogs that retain one or moreacetylenic portions, thus providing still more additional analogs. Allanalogs are considered part of the present invention and are herebyexplicitly incorporated herein. Separation and identification of thecompounds can be accomplished by methods known in the art (TLC, HPLC,GC, etc.)

Retention of acetylenic portions within the resolvin analog isconsidered to be advantageous. The synthesis can be shortened (thehydrogenation step or steps can be eliminated or monitored so that onlyselective hydrogenation occurs). The resultant acetylenic containingresolvin compounds retain similar bioactivies to the corresponding fullyhydrogenated olefinic containing resolvins. Additionally, it is believedto be advantageous to avoid hydrogenation of those olefinic bonds thatare generated from acetylenic portions which correspond to “cis”configurational isomers with respect to naturally occurring DHA and EPAcompounds. That is, retrosynthetically, it is advantageous to prepareDHA and EPA compounds having acetylenic portions where previously cisdouble bonds existed in the molecule.

For example, Scheme I provides for the general preparation of one classof resolvins

It should be noted, for example, that the “R” groups are used asinhibitors for blocking oxidation of the 7-OH and/or 17-OH, formingketones at the C-7 or C-17 position(s).

In synthetic scheme II, the synthesis of the 7(8)-methano-analog isbased on the biotemplate of the epoxide intermediate in the biosynthesisof bioactive products generated by exudates and cell from DHA as theprecursor. In this example, Pd⁰/Cu¹ catalyzed coupling of the vinylbromide acetylene with the alkynyl alcohol proceeds after brominationand phosphate production to give a phosphonate as an intermediate. Thephosphonate is subject to condensation of the lithio derivate with thealdehyde to yield a mixture of Δ9-10 cis(E)(Z) trans isomers. These canbe converted by treatment with catalytic amounts of iodine. To protectthe triple bonds, the silyl protecting group at carbon 17 is replaced byacetate. Lindlar catalytic reduction isused to reduce the triple bondsin quinoline. The product(s) are deacetylated to give the stablecyclopropyl 7(8) methano analog of the equivalent labile epoxide analogthat is involved in resolvin biosynthesis of exudates in vivo and/or incells, such as microlial cells of the brain or human leukocytes.

These analogs are important therapeutics because in addition to actingat the site/receptor for 7,8,17R-triHDHA resolvin as a mimetic to stopleukocyte recruitment as an agonist to stimulate/promote resolution andto pharmacologically inhibit inflammation, it also serves as aninhibitor of enzymes in vivo. Thus the compounds inhibit proinflammatorylipid mediators such as leukotrienes and also lead to an accumulation insitu of upstream resolvins in the biosynthetic pathway (See FIG. 8).Thus, these class of compounds serve a dual purpose: mimetic of resolvin7,8,17-tri-HDHA and as a substrate level inhibitor.

Scheme VI represents another class of compounds, where again,“protection” of the potentially oxidizable 5 and/or 18 hydroxyls toketones. Use of “R” groups, as described herein, provides the ability toprevent the oxidation, and therefore the bioavailability of thebioactive compound.

The analogs within synthetic scheme VI can be prepared by coupling thevinyl bromide as prepared in K. C. Nicolaou et al. Angew. Chem. Int. Ed.Engl 30 (1991) 1100-1116) and coupled using Pd/Cu coupling chemistry.The resultant intermediate can be selectively hydrogenated with theLindlar catalyst and hydrogen to produce various acetylenic products, aswell as penatene containing products. Deprotection of the alcohols andconversion to carboxylic acids, esters, etc. can be accomplished byknown methods.

Scheme for 10,17 diHDHA Analogs

10,17 diHDHA's are depicted in FIG. 8 and are of interest because thebiosynthesis of 10,17 di-HDHA differs from the other compounds of FIG.8. It is produced via 15-lipoxygenase action on DHA (pH of about 8.5)under conditions that favor hydroperoxidation at the 17 position of DHAwhich is then converted into the 16,17 epoxide. The 16(17) epoxidecarries the conjugated triene chromophore and opens via a carboniumcation intermediate with OH attack at the 10 position to affor10,17-diHDHA. Human tissues and isolated cells produce this via the15-lipoxygenase as well as additional enzymes. This compound has beenprepared by using soyean 15-lipoxygenase with DHA as the substrate at apH of about 8.5, presented in micelle configuration. The 10,17 di-HDHAwas isolated using RP-HPLC as described herein. It was found that the10,17 di-HDHA inhibited both PMN migration into the peritoneum (zymosaninduced peritonitis) of mice given Zymosan and inflammation. Hence,protection at the C-10 hyroxyl position with an “R protecting group”should prevent metabolic conversion and incrase stability and activationto block PMN infiltration and acute inflammation.

Scheme for 4,11,17-triHDHA

Scheme for 5,18 di-HEPA

The preparation of 5,18-diHEPA analogs is achieved using a conjugatedaddition of a vinyl zirconium reagent 3-(1-octen-1-yl)cyclohexanone asin Sun, R. C., M. Okabe, D. L. Coffen, and J. Schwartz. 1992. Conjugateaddition of a vinylzirconium reagent: 3-(1-octen-1-yl)cyclopentanone(cyclopentanone, 3-(1-octenyl)-, (E)-). In Organic Syntheses, vol. 71.L. E. Overman, editor. Organic Syntheses, Inc., Notre Dame, Ind. 83-88using Schwartz's reagent as prepared in Buchwald, S.L., S.J. LaMaire,R.B. Nielsen, B.T. Watson, and S.M. King. 1992. Schwartz's reagent(zirconium, chlorobis(h5-2,4-cyclopentadien-1-yl)hydro-). In OrganicSynthesis, vol. 71. L.E. Overman, editor. Organic Syntheses, Inc., NotreDame, Ind. to constructthe zirconiated intermediate. Treatment withDIBAL as in Ishiyama, T., N. Miyaura, and A. Suzuki. 1992.Palladium(0)-catalyzed reaction of 9-alkyl-9-borabicyclo[3.3.1]nonanewith 1-bromo-1-phenylthioethene:4-(3-cyclohexenyl)-2-phenylthio-1-butene. In Organic Syntheses, vol. 71.L.E. Overman, editor. Organic Syntheses, Inc., Notre Dame, Ind. providesthe di-HEPA ring containing analog. It should be understood that thecyclohexanone reagent can be substituted with any number ofsubstituents, thereby providing the resultant substituted orunsubstituted aromatic ring within the di-HEPA analog.

Again, it should be noted that inclusion of an “R protecting group” atC-5 and/or C-18 positions helps to inhibit oxidation of the hydroxylgroup to a ketone. Additionally, it is believed that the use of a ringwithin the structure helps to contrain confirmation about the moleculeand affects receptor ligand interaction(s).

Scheme for 7,17 Resolvin Ring Containing Analogs

Scheme for 7,8,17-Trihdroxy-HDHA with Ring Structure

Scheme for 7,16,17-Tri-HDHA Ring Analogs

Results Lipidomics of the Exudate Resolution Phase

It is well appreciated that orderly resolution in healthy individuals isinfluenced by both systemic and local host factors that includenutrition, metabolic status (i.e., diabetes is associated with delayedhealing) and circulatory status as some of the key determinants in theduration of resolution (39). In experimental acute inflammatorychallenge that undergoes spontaneous resolution, namely in the murineair pouch model of exudate formation and resolution, it was found atemporal dissociation between the formation and actions of localchemical mediators (10). Leukotrienes and prostaglandins are generatedrapidly and appear with leukocyte recruitment to the air pouch exudatein line with their known actions as proinflammatory mediators. Lipoxinbiosynthesis concurs with spontaneous resolution and the loss of PMNfrom the murine air pouch exudate, providing evidence that functionallydistinct lipid mediator profiles switch from proinflammatory toanti-inflammatory mediators such as lipoxins during resolution (10).

Recently, it was found that EPA is transformed in murine exudatestreated with ASA to novel products that possess anti-inflammatoryproperties, providing a potential mechanism for omega-3 beneficialactions in many diseases (2). Since DHA is cardioprotective (22),abundant in brain and retina and displays an impact in many physiologicprocesses (28-32), lipidomic analyses were undertaken to determinewhether inflammatory exudates utilize DHA in the resolution phase withASA treatment.

FIG. 9 depicts the approach developed to isolate, examine, characterizeand separate the various components of the exudates. Until now, it wasunappreciated how many different compounds are generated through thebiochemical pathway. Each compound is unique and precise separation andcharacterization was required to isolate each component. In general, asample of the exudates is extracted and then separated into componentsvia solid phase extraction followed by chromatography and mass spectralanalysis. GC-MS can also be employed to help identify separatecomponents. UV analysis is also often helpful. The physical propertiesof the compounds are then identified and placed into a library todetermine which compounds are unique and previously unknown. Furtherstructural elucidation is undertaken (NMR, MS/MS, IR, etc.) prior toscale up. Production of the compounds can be accomplished via biogenicsynthesis and or traditional organic synthesis, such as provided herein.

Inflammatory exudates obtained within the resolution phase formed withindorsal skin air pouches following injection of TNFα, DHA and aspirintreatment contained several previously unknown novel compounds revealedwith LC-MS-MS analysis (FIG. 1). It is noteworthy that the Lab Diet 5001used to feed these mice contained 1.86% DHA and 1.49% EPA with <0.25%arachidonic acid (Purina Mills). Additional mass spectral analysisemploying both GC-MS (with derivatized products) and LC-UV-MS-MS-basedanalyses (which did not require derivatization) indicated that theinflammatory exudate-derived materials contained novel hydroxy acidsproduced from both DHA and EPA, namely these products were not known asreported lipid mediators. The EPA-derived products were recentlyestablished (2). Selected ion chromatograms and MS-MS from resultsacquired at m/z 343 were consistent with the production of 17-HDHA (FIG.1, Panels A and B), with lesser amounts of 7S- and 4S-HDHA (FIG. 1,Panel A) within the exudates. These products co-eluted with authentic17(R/S racemic)-HDHA and 4S-HDHA (qualified by NMR; see Methods) in 3different chromatographic systems (not shown), and their basicstructural properties were consistent with those of related DHA-derivedproducts (cf. 28, 29, 30). ASA-treatment also gave novel di- andtri-hydroxy products carrying the DHA backbone within the inflammatoryexudates; at this dose ASA completely inhibited the in vivo productionof thromboxane and prostanoids. Importantly, ASA treatment in vivo andCOX-2 gave previously unknown products from DHA that possess bioactiveproperties (vide infra).

Results in FIG. 1, Panel C show the MS-MS spectra of adihydroxy-containing DHA with fragment ions consistent with thestructure shown in the inset, namely 7,17-diHDHA; m/z 359 [M-H], m/z 341[M-H—H₂O], m/z 323 [M-H-2H₂O], m/z 315 [M-H—CO₂], and m/z 297[M-H—CO₂—H₂O]. Additional diagnostic ions consistent with the 7- and17-hydroxy-containing positions were present at m/z 261, 247, and 217. Arepresentative of the several novel trihydroxy-containing DHA-derivedcompounds also present in inflammatory exudates is shown in FIG. 1,Panel D. Ions present were consistent with its [M-H]=m/z 375, 357[M-H—H₂O], 339 [M-H-2H₂O], 331 [M-H—CO₂], 313 [M-H—CO₂—H₂O], 306, 303,276, 273, 255 [273-H₂O], 210, 195, and 180. These physical properties(i.e. MS-MS, UV, LC retention time) were used throughout to identifythese and related compounds and to assess their bioimpact. Thesecompounds were deemed of interest because transfer of materialsextracted from inflammatory exudates in DHA plus ASA pouches to naïvemouse (i.v. or via i.p. administration) sharply reduced zymosan-inducedPMN infiltration by ˜60%, indicating the in vivo utilization of DHA andproduction of bioactive products within exudates (vide infra).

The Role of COX-2 and ASA in Biosynthesis of R-Containing HDHA

Chirality of the alcohol group at carbon-17 (FIG. 1B) was establishedfor the product that matched exudate-derived 17-HDHA using a chiral HPLCcolumn. The alcohol at carbon 17 position proved to be predominantly inthe R configuration (>95%; n=4), indicating that this was indeed a novelproduct of enzymatic origin formed in vivo that was not known earlier.For example, 17S-HDHA is generated via 15-lipoxygenation or viaautooxidation in racemic 50:50 ratio of R/S mixtures cf. (40, 41).Hence, the presence of the alcohol group in the R configuration as17R-HDHA from exudates (FIG. 1) was indicative of an enzymatic origin.The substrate channel of COX-2 is larger than COX-1 (26), suggesting thepossibility of substrates larger than arachidonic acid. Consistent withthis, DHA was transformed by rhCOX-2 to 13-HDHA (FIG. 2; left panel).The MS-MS obtained were consistent with oxygen addition at the 13position (i.e., m/z 193 and m/z 221) and, when COX-2 was treated withaspirin to acetylate serine within the catalytic site (1, 26, 42), DHAwas enzymatically converted to 17R-HDHA (FIG. 2; right panel). The MS-MSand diagnostic ions at m/z 343 [M-H], 325 [M-H—H₂O], 299 [M-H—CO₂] 281[M-H—H₂O—CO₂], 245 and 274 consistent with 17-carbon alcohol group andchiral analysis using chiral HPLC with reference materials (see Methods)indicated that the conversion of DHA by aspirin-acetylated COX-2 yieldedpredominantly (>98%) 17R-HDHA. The product of COX-2 matched the physicalproperties of the dominant 17-hydroxy-containing DHA-derived compoundidentified in inflammatory exudates (FIG. 1) in vivo with aspirintreatment. Unlike cyclooxygenase-1, shown earlier not to convert DHA(43), results with recombinant COX-2 in Table 1 and FIG. 2 indicate thataspirin treatment of this enzyme generates predominantly 17R-HDHA. Othercommonly used nonsteroidal anti-inflammatory drugs, i.e. indomethacin,acetaminophen, or the COX-2 inhibitor (e.g. NS-398), did not giveappreciable amounts of 17R-HDHA. Treatment with aspirin gave areciprocal relationship between 17 versus 13-position oxygenation. Alsoin these incubations, indomethacin, acetaminophen, and NS-398 eachreduced the overall oxygenation of DHA [to 13- as well as 17-HDHA (seeTable 1)], but did not share the ability of ASA to produce 17R-HDHA. Fordirect comparison, conversion of C20:4 by ASA-acetylated COX-2 to15R-HETE (67%±5% substrate conversion; n=3) was monitored in parallelwith the conversion of DHA to 17R-HDHA (52±3%; mean±S.E.M.; n=3).

Brain and Vascular Biosynthesis of the New Compounds

In brain, COX-2 is present in constitutive as well as in induciblepool(s) (28, 44). Results in FIG. 3 (A and B) indicate thataspirin-treated brain contained 17R-HDHA produced from the endogenoussources of DHA. To address the possible cell types involved in 17R-HDHAgeneration in brain, human microglial cells were exposed to TNFα, whichup-regulated expression of COX-2, followed by treatment with ASA and theagonist ionophore A23187. Human microglial cells generated 17R-HDHA inan ASA-dependent fashion (FIG. 3C). Hypoxia is also known to induceCOX-2 (45), and hypoxic endothelial cells exposed to cytokine IL-1β, asendothelial cells might encounter at inflammatory loci or with ischemicevents (23), treated with aspirin were a source of 17R-HDHA (FIG. 4).

Of interest, in the absence of ASA treatment, 17S-HDHA and corresponding17S-hydroxy-containing diHDHA and triHDHA were products in murineexudates and human cells. Their formation differs from the presentbiosynthesis in that, rather than COX-2-ASA, 15-lipoxygenase initiatesbiosynthesis in sequential lipoxygenation reactions to produce di- andtrihydroxy-DHA (i.e., 7S,17S-diHDHA, 10,17S-diHDHA, 4S,17S-diHDHA,4S,11,17S-triHDHA and 7S,8,17S-triHDHA; cf. Table 2) via epoxideintermediates.

Bioactions of the New Compounds

Since microglial cells are involved in host defense and inflammation inneural tissues, human microglial cells were incubated with the COX-2products 13- and 17R-HDHA (each at 100 nM) to determine if they had animpact on the generation of inflammatory mediators (FIG. 5A inset). AtnM concentrations, these novel cyclooxygenase-2 products inhibitTNFα-induced cytokine production with apparent IC₅₀˜50 pM, as did the17-containing di- and trihydroxy-HDHA compound (FIG. 5A). Next, the HDHAwere tested for their ability to regulate transendothelial migration ofhuman PMN. In the nM range, neither of the COX-2-derived monohydroxy-DHAproducts had a direct impact on PMN transmigration across endothelialcell monolayers (FIG. 5B). This finding contrasts results obtained withboth EPA- and arachidonic acid-derived eicosanoid products that directlydownregulate PMN transmigration in vitro (2, 3). For purposes of directcomparison, results with an ASA-triggered EPA pathway product18R,5,12-triHEPE (P<0.01 by ANOVA) (cf. Ref. 2) were compared to thoseobtained with 15-epi-16-para(fluoro)-phenoxy-lipoxin A₄ (P<0.01 byANOVA), a stable analog of aspirin-triggered 15R-lipoxin A₄ producedwith aspirin treatment from arachidonic acid (10, 37).

Biosynthesis of Novel Docosanoids by Human PMN: Cell-Cell InteractionProducts Matched in Exudates

Next, since PMN interact with vascular cells during inflammation (7),the contribution of leukocytes was assessed in the production of thenovel di- and tri-hydroxy compounds present in inflammatory exudates(FIG. 1A-D). To this end, human PMN were exposed to zymosan and 17R-HDHAproduced via ASA-treated COX-2 or endothelial cells. PMN engaged inphagocytosis transform 17R-HDHA to both di- and tri-hydroxy-DHA (FIG.6). The main conversions were to dioxygenation products including7S,17R-diHDHA and 10,17R-diHDHA with lesser amounts of 4S,17R-diHDHA asthe main dihydroxy-containing products present when monitored at m/z 359(see FIG. 6). In addition, novel 17R-trihydroxy-containing productsmonitored at m/z 375 were present including 4S,11,17R-triHDHA as well asa set of trihydroxytetraene containing 7,8,17R-triHDHA (see Table 2).These compounds formed by human PMN match those produced within exudatesgenerated in vivo in both their chromatographic behavior and prominentions present in their respective mass spectra. LC-MS and on-line UVdiode array profiles shown in FIG. 7 from exudates of mice treated withASA indicate the in vivo production of both sets of 17R series di- andtri-hydroxy-containing products that carry triene and tetraenechromophores (see Table 2). Sources for these trihydroxy-DHA products asschematically illustrated include omega-1 hydroxylation at carbon 22 ofeither 7S,17R-diHDHA or 4S,17R-diHDHA via a p450-like reaction (see Ref.6 and FIG. 8) that likely represents inactivation pathway products aswith leukotrienes such as formation of 20-OH-LTB₄, a product ofomega-oxidation of LTB₄ (2, 3, 6).

Transformation of 17R-HDHA by activated human PMN involved a5-lipoxygenase and an LTA₄ synthase reaction that gave triHDHA productsvia the formation of respective 4S-hydro(peroxy)-17R-hydroxy- and7S-hydro(peroxy)-17R-hydroxy-containing intermediates. Each wasconverted to epoxide-containing intermediates (i.e. 4(5) epoxide or 7(8)epoxide intermediates) that open via hydrolysis to give rise to4S,11,17R-triHDHA or in a parallel route to diols such as thetrihydroxytetraene set 7S,8,17R-triHDHA (see FIGS. 4, 7 and 8). Themechanism used by PMN to convert the 17R-HDHA precursor appeared similarto that established and identified for the epoxide generating capacityof the human PMN 5-lipoxygenase, which performs both a lipoxygenationand epoxidation step (2, 4) as demonstrated with the potato5-lipoxygenase (46). The conversion of 17R-HDHA by human PMN displayedsimilar features as those established for the conversion of arachidonicacid to either leukotriene B₄ or lipoxins as well as the recentlyuncovered 18R series of EPA products (4, 9). These were modeled in vivobiosynthetic sequences of events using both plant (5-LO potato or 15-LOsoybean) and human enzymes added in tandem “one pot” incubations thatproduce these compounds and matched those of human PMN and murineexudates (see Methods and Table 2). The findings with 17R-HDHA are ofinterest because the S isomer 17S-HDHA, a product of 15-lipoxygenase,can inhibit human neutrophil 5-lipoxygenase production of leukotrienesfrom endogenous substrate (47). Along these lines, it was found that17R-HDHA was converted by isolated potato 5-LO to both4S-hydro-(peroxy)-17R-hydroxy- and7S-hydro-(peroxy)-17R-hydroxy-containing products that were reduced to4S,17R-diHDHA and 7S,17R-diHDHA, respectively. These, as well astrihydroxy-DHA (see Table 2), are new products and indicate that17R-HDHA is a substrate for 5-lipoxygenase and its epoxidase activity.The biogenic synthesis and physical properties of the compounds produced(i.e. major ions of the methyl ester trimethylsilyl-derivatives) withGC-MS analysis were consistent with the fragments obtained withoutderivatization using LC-MS-MS (see Table 2) and support the proposedstructures as well as was both the murine exudate and human PMNbiosynthesis from DHA (cf. 29 for monohydroxy products). Of interest,when added to human PMN, 17R-HDHA prevented formation of leukotrienesboth in vitro with human PMN and in murine exudates (n=4; not shown).

Inhibition of PMN Recruitment in Peritonitis and Air Pouch:Anti-Inflammatory Properties (i.v. and Topical) of Resolvins

Although 17R-HDHA did not directly inhibit neutrophil transmigration invitro (FIG. 5B), 17R-HDHA did regulate in vivo PMN exudate cell numbersin peritonitis as well as in the dermal air pouch (FIG. 5C). Also,17R-HDHA was a potent inhibitor of zymosan-induced peritonitis, as wereboth the di- and tri-hydroxy-containing compounds (i.e. 7S,17R-diHDHAand 4S,11,17R-triHDHA). In addition to their ability to inhibit PMNrecruitment when injected i.v. within zymosan-induced peritonitis, the17R-hydroxy-HDHA-derived di- and trihydroxy-containing products provedto be potent regulators of leukocyte recruitment into the air pouch whenadministered i.v. as well as topically with local administration (FIG.5C). Thus, the present results indicate that human and murine leukocytesconvert 17R-HDHA to a novel series of 17R-hydroxy-containing di- andtriHDHA; namely, an ASA-triggered circuit to utilize DHA to produce a17R-series of docosanoids (FIG. 8).

The present results indicate that cells expressing cyclooxygenase-2 inexudates and brain treated with aspirin enzymatically transform omega-3DHA to previously unrecognized compounds with bioactive properties ininflammation-resolution, i.e. a novel 17R series of di andtri-hydroxy-docosahexaenoic acids. The ASA-acetylated COX-2 present inthese tissues generates predominantly 17R-HDHA that is converted furtherenzymatically to potent bioactive 17R series via lipoxygenation andepoxidation in leukocytes to both di- and tri-hydroxy-containing noveldocosanoids (see FIG. 8). DHA is the most unsaturated of the omega-3polyene family of fatty acids in mammalian and fish tissues. In humans,DHA is abundant in brain, retina, and testes (28, 48). The levels of DHAincrease in adult human brain with age, which is required for optimalneural development (49) and DHA is rapidly esterified in retinalepithelium photoreceptors as well as into the phospholipids of restinghuman neutrophils (28, 50). At high micromolar values, DHA is held topossess both physiologic roles and direct action on neural voltage gatedK⁺ channels (51), binds RXR in neural tissues (52) and is held to be theactive compound of fish oil supplements that is cardioprotective (21).Also, addition of DHA can correct and reverse the pathology associatedwith cystic fibrosis in cftr−/− mice (53). However, it is not clear fromthe results of these studies (21, 51, 52) or of the many reportedclinical trials whether DHA is precursor to potent bioactive structuresthat are responsible for the many reported properties attributed to DHAitself in regulating biological systems of interest.

The three major lipoxygenases (i.e. 5-LO, 12-LO, and 15-LO) that act onarachidonate can each convert DHA to S-containing products, but theirfunction in the immune system or elsewhere is not clear. In the brain,12-lipoxygenase of pineal body converts DHA to 14S-HDHA and15-lipoxygenase to 17S-HDHA (40). DHA can also be converted by humanneutrophils to 75-HDHA that does not stimulate chemotaxis (31), andretina converts DHA to both mono- and di-hydroxy products vialipoxygenase(s) (28). While not a substrate for COX-1 (43), oxidizedisoprostane-like compounds can also be produced from DHA that appear toreflect oxidative free radical catalyzed events (54). Hence, the new17R-hydroxy series of docosanoids generated by neural tissues,leukocytes, and inflammatory exudates uncovered in the presentexperiments and their role(s) are of interest ininflammation-resolution, a process now considered to be associated withmany human diseases.

Although omega-3 fish oils encompassing both EPA and DHA could have abeneficial impact in the treatment of many chronic diseases (such ascardiovascular disease, atherosclerosis and asthma, as well asanti-tumor and anti-proliferative properties (15, 55)), the molecularrationale for their use remains of interest. Most of the earlier studiesfocused on uptake of omega-3 PUFA (i.e. EPA and DHA), namely theiresterification into phospholipid and other lipid stores of many humantissues that in some cells reduces the availability of endogenousarachidonic acid for processing to pro-inflammatory prostaglandins (55).The body of results now available indicates that, in addition topro-inflammatory roles, specific 15-lipoxygenase, 5-LO and/or LO-LOinteraction products formed during cell-cell interactions such aslipoxins serve as endogenous anti-inflammatory mediators promotingresolution (9, 10, 12). Like other lipoxygenase-derived eicosanoids,lipoxins are potent-local acting in subnanomolar levels with precisestereochemical requirements for evoking their actions (4, 9). Hence, theproduction of 18R and 15R series products from EPA that inhibit PMNtransmigration and inflammation within the low nanomolar rangeemphasizes the functional redundancies within chemical mediatorsproduced from the omega-3 family of polyene fatty acids, namely therecently identified compounds from COX-2 EPA (2) or DHA-derivedcompounds as indicated from the present results (FIGS. 6-8). It isimportant to note that with these small molecular weight mediatorssubtle changes in chirality of alcohol—i.e. S to R—can change a compoundfrom active to inactive or vice versa (3, 4, 9). In this regard, the15R-hydroxy-containing compounds generated from either arachidonic acidor EPA and 18R series from EPA, as well as 17R-hydroxy series from DHA,each display similar functional redundancies in inflammation-resolution.Hence, uncovering the 17R series of both mono- and di-oxygenationproducts in inflammatory exudates and a role for COX-2 in the generationof the 17R-hydroxyl configuration in HDHA described here for the firsttime opens new avenues for considering the overall functionalredundancies of mediators that dampen and/or counter the manypro-inflammatory signals to promote resolution.

Cyclooxygenase-2 is induced in most inflammatory cell types, but canalso be constitutive in neural and vascular tissue (44, 56). Theimportance of the enlarged substrate tunnel in cyclooxygenase-2 becomesof interest when considering possible physiologic roles of this enzymein these localities in vivo. It is now clear from numerous studies thataspirin has beneficial effects in and apart from other nonsteroidalanti-inflammatory drugs (57, 58). In this regard, aspirin has a uniqueability to acetylate both isoforms of cyclooxygenase (COX-1 and COX-2).It is also noteworthy that DHA is cardioprotective in the ischemic heart(22) and that COX-2 is involved in preconditioning (19) as well asresolution (12). The present invention provides that DHA is a precursorand is converted to 17R-HDHA via aspirin-acetylated COX-2 at sites ofinflammation in vivo (FIG. 1), murine brain (FIG. 3), and by acetylatedrecombinant COX-2 in vitro (FIG. 2). Both 13- and 17R-HDHA inhibitcytokine generation by microglial cells at the transcript level in thepicomolar range (FIG. 5A). Human microglial cells generate these17R-HDHA series products when given aspirin and TNFα, which upregulateCOX-2 expression (FIG. 3C). In addition, murine inflammatory exudatesproduced a family of novel di- and tri-hydroxy products that were alsoproduced by human PMN via transcellular processing of 17R-HDHA. Theproposed pathways for transcellular processing of acetylatedCOX-2-derived 17R-HDHA highlighting the generation of dioxygenatedintermediates and epoxidation to form novel diHDHA during vascularinflammation-associated events are illustrated in FIG. 8.

It should be noted that these and related structures can be generatedvia cell-cell interactions or single cell types as depicted in FIG. 8,but could in theory also be produced via several sequential oxygenationroutes by a single enzyme as well (see FIG. 8 legend). When theseproducts were prepared by biogenic total synthesis and added back viatopical administration into the air pouch, they inhibited TNFα-inducedleukocyte infiltration. Also, with i.v. administration these compoundsinhibited leukocyte recruitment in both murine air pouch and inzymosan-induced peritonitis (FIG. 5). Taken together, these resultsindicate that aspirin-acetylated COX-2-derived products can downregulatecytokine generation and leukocyte (i.e., neutrophil) recruitment tosites of inflammation. The EPA-derived 5,12,18R series product proved tobe as effective as a potent stable analog of 15-epi-lipoxin A₄ inpreventing leukocyte diapedesis and exudate formation (see FIG. 5C).Since 17R-HDHA did not have a direct impact on human PMN transmigrationin these conditions, but reduced exudate PMN numbers in vivo as well asregulates gene expression in human microglial cells, it is highly likelythat a multi-level mechanism of action accounts for the in vivoproperties of this ASA-triggered pathway. Moreover, there appear to befunctional redundancies between the pathways in that the 18R series fromEPA- and 17R series DHA-derived hydroxy-containing compounds share intheir ability to regulate PMN exudate numbers (FIG. 5).

Emergence of the finding that arachidonic acid-derived lipoxins inhibitPMN trafficking and serve as endogenous anti-inflammatory mediatorswhile activating monocytes in a nonphlogistic fashion (11, 59), as wellas accelerating the uptake of apoptotic PMN by macrophages at sites ofinflammation (28), indicates that not all lipoxygenase pathway productsfrom the arachidonic acid precursor are “pro”-inflammatory. Given theirlonger half-life and bioavailability, the metabolically more stableanalogs of these local-acting lipid mediators derived from arachidonatein vivo and prepared by total organic synthesis provide further evidencefor their roles in promoting resolution (37). Moreover, these resultssuggest that the new resolving properties belong to a larger class ofendogenous compounds with mechanisms directed towards enhancingresolution. Also, the link between anti-inflammation and enhancedendogenous antimicrobial activities (13) by lipoxins andaspirin-triggered lipoxins sets a unique precedent for the importance ofcell-cell communication and transcellular biosynthesis in host defenseand in the clearance and resolution of inflammatory sequelae.

The present results disclosing 17R series oxygenated DHA products andwith the 15R and 18R series from eicosapentaenoic acid as prototypes(2), taken together, suggest that the generation of local-acting lipidmediators with beneficial actions relevant in human disease may not berestricted to arachidonic acid alone as an important precursor. Also,they indicate that transcellular biosynthesis unveils previouslyunrecognized pathways that are evoked by aspirin treatment with DHA.Acetylated COX-2 acts in an “R-oxygenation” mechanism to initiate theconversion of DHA to a 17R series of di- and tri-hydroxy docosanoidsthat display downregulatory actions in vivo in inflammation as do theomega-3 EPA-derived 18R-series-products. Hence, it follows that, onceinflammation is initiated, upon aspirin treatment with omega-3supplementation, these pathways can be operative in vascular tissues togenerate products that appear to have properties as aspirin-triggeredlipid mediators similar to those of either the 15-epi-lipoxins or 18R-and 15R series products from EPA. These compounds are generated vialipoxygenation followed by epoxidation and subsequent steps (FIG. 8 andcf. Ref. 2). Also of interest are the findings that, in the absence ofaspirin, COX-2 converts DHA to 13-HDHA, a previously unknown route thatmight also be relevant in tissues that constitutively express COX-2,which is also converted to dihydroxy DHA products (4,13-diHDHA,7,13-diHDHA, and 13,20-diHDHA), and during resolution, induction andconversion by 15-lipoxygenase (10) to 10,17S-diHDHA and 7S,17S-diHDHA(See FIGS. 10, 11, 12, and 14).

Since the properties of the omega-3-derived products from acetylatedCOX-2 via transcellular biosynthesis appear to dampen events ininflammation apparently in a functionally redundant fashion (i.e.17R-HDHA series, 18R- and 15R-HEPA series), the term “Resolvins” isintroduced for this family of new compounds and bioactions. Resolvins,by definition, are endogenously generated within the inflammatoryresolution phase and downregulate leukocytic exudate cell numbers toprepare for orderly and timely resolution. The present results indicatethat the 17R series of di- and trihydroxy DHA pathways are potent inmodels relevant in inflammation. It is likely that these compounds willalso have actions in other tissues, in view of the many reports of theclinical actions for EPA and DHA, where high concentrations of thesePUFA are used and required to evoke responses in vitro. The presentresults indicate that cell-cell interactions at sites ofinflammation-resolution utilize omega-3 fatty acids to generate novelomega-3-derived products including 17R-HDHA series and 18R-HEPE seriesof oxygenated bioactive products termed Resolvins (FIGS. 8 and 13).Given their potent actions, the production of Resolvins may, in part,provide a rationale underlying the beneficial actions of omega-3 fattyacids (15-22) in chronic immune and vascular diseases as well as serveas new biotemplates for therapeutic development.

TABLE 1 Impact of NSAIDs on COX-2 conversion of DHA % Increase of NSAID% Inhibition of 13-HDHA 17-HDHA ASA 85.7 ± 5.6% 97.8 ± 2.0% Indomethacin89.8 ± 0.5% 0.0% Acetaminophen 87.7 ± 4.3% 0.0% NS398 (COX-2 inhibitor)97.3 ± 1.0% 0.0%

Results are the mean±SEM, n=3. Products were extracted, identified, andquantitated using deuterium internal standards and LC-MS-MS (seeMethods). NSAIDs were incubated 30 min with human recombinant COX-2 (seeMethods); ASA [2 mM], indomethacin [200 μM], acetaminophen (500 μM), andNS398 (100 μM).

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Although the present invention has been described with reference topreferred embodiments, persons skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention. All publications and references citedherein, including those in the background section, are expresslyincorporated herein by reference in their entirety.

What is claimed is:
 1. A method of treating or preventing inflammationin a subject, comprising administering to the subject a compound havinga structure of the formula:

wherein P₁, P₂ and P₃, if present, each individually is ahydroxyl-protecting group or hydrogen atom; R₁, R₂ and R₃, if present,each individually is a hydrogen atom, substituted or unsubstituted,branched or unbranched alkyl group, substituted or unsubstituted arylgroup, substituted or unsubstituted, branched or unbranched alkylarylgroup, or halogen atom; Z is —C(O)OR^(d), —C(O)NR^(c)R^(c), —C(O)H,—C(NH)NR^(c)R^(c), —C(S)H, —C(S)OR^(d), —C(S)NR^(c)R^(c), or —CN; eachR^(a) is independently selected from hydrogen, (C1-C6) alkyl, (C3-C8)cycloalkyl, cyclohexyl, (C4-C11) cycloalkylalkyl, (C5-C10) aryl, phenyl,(C6-C16) arylalkyl, benzyl, 2-6 membered heteroalkyl, 3-8 memberedcycloheteroalkyl, morpholinyl, piperazinyl, homopiperazinyl,piperidinyl, 4-11 membered cycloheteroalkylalkyl, 5-10 memberedheteroaryl, and 6-16 membered heteroarylalkyl; each R^(b) is a suitablegroup independently selected from ═O, —OR^(d), (C1-C3) haloalkyloxy,—OCF₃, ═S, —SR^(d), ═NR^(d), ═NOR^(d), —NR^(c)R^(c), halogen, —CF₃, —CN,—NC, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)R^(d), —S(O)₂R^(d),—S(O)₂OR^(d), —S(O)NR^(c)R^(c), —S(O)₂NR^(c)R^(c), —OS(O)R^(d),—OS(O)₂R^(d), —OS(O)₂OR^(d), —OS(O)₂NR^(c)R^(c), —C(O)R^(d),—C(O)OR^(d), —C(O)NR^(c)R^(c), —C(NH)NR^(c)R^(c), —C(NR^(a))NR^(c)R^(c),—C(NOH)R^(a), —C(NOH)NR^(c)R^(c), —OC(O)R^(d), —OC(O)OR^(d),—OC(O)NR^(c)R^(c), —OC(NH)NR^(c)R^(c), —OC(NR^(a))NR^(c)R^(c),—[NHC(O)]_(n)R^(d), —[NR^(a)C(O)]_(n)R^(d), —[NHC(O)]_(n)OR^(d),—[NR^(a)C(O)]_(n)OR^(d), —[NHC(O)]₇,NR^(c)R^(c),—[NR^(a)C(O)]_(n)NR^(c)R^(c), —[NHC(NH)]_(n)NR^(c)R^(c) and—[NR^(a)C(NR^(a))]_(n)NR^(c)R^(c); each R^(c) is independently aprotecting group or R^(a), or, alternatively, each R^(c) is takentogether with the nitrogen atom to which it is bonded to form a 5 to8-membered cycloheteroalkyl or heteroaryl which may optionally includeone or more of the same or different additional heteroatoms and whichmay optionally be substituted with one or more of the same or differentR^(a) or suitable R^(b) groups; each R^(d) independently is a protectinggroup or R^(a); X, if present, is a substituted or unsubstitutedmethylene, an oxygen atom, a substituted or unsubstituted nitrogen atom,or a sulfur atom; Q represents one or more substituents and each Qindividually, if present, is a halogen atom or a branched or unbranched,substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl,alkoxy, aryloxy, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,aryloxycarbonyl, amino, hydroxy, cyano, carboxyl, alkoxycarbonyloxy,aryloxycarbonyloxy, or aminocarbonyl group; U, if present, is a branchedor unbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl,cycloalkyl, aryl, alkoxy, aryloxy, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aryloxycarbonyl, alkoxycarbonyloxy, oraryloxycarbonyloxy group; and each n independently is an integer from 0to 3, wherein any carbon-carbon double bond may be either cis or trans;or a pharmaceutically acceptable salt thereof.
 2. A method of claim 1,wherein the compound has a structure of the formula:

or a pharmaceutically acceptable salt thereof.
 3. A method of claim 1,wherein the compound has a structure of the formula:

wherein any carbon-carbon double bond may be either cis or trans, or apharmaceutically acceptable salt thereof.
 4. A method of claim 1,wherein the compound has a structure of the formula:

wherein any carbon-carbon double bond may be either cis or trans, or apharmaceutically acceptable salt thereof.
 5. A method of claim 4,wherein the compound has a structure of the formula:

wherein Z is —C(O)OR^(d), R^(d) is a protecting group or R^(a), R^(a) ishydrogen, (C1-C6) alkyl, (C3-C8) cycloalkyl, cyclohexyl, (C4-C11)cycloalkylalkyl, (C5-C10) aryl, phenyl, (C6-C16) arylalkyl, benzyl, 2-6membered heteroalkyl, 3-8 membered cycloheteroalkyl, morpholinyl,piperazinyl, homopiperazinyl, piperidinyl, 4-11 memberedcycloheteroalkylalkyl, 5-10 membered heteroaryl or 6-16 memberedheteroarylalkyl, and P₁ and P₂ each individually are a hydrogen atom ora hydroxyl-protecting group, and wherein any carbon-carbon double bondmay be either cis or trans, or a pharmaceutically acceptable saltthereof.
 6. The method of claim 5, wherein the carbon atom at C-17 hasan R configuration.
 7. The method of claim 5, wherein the C-10 carbonatom has an S configuration and the carbon atom at C-17 has an Sconfiguration.
 8. The method of claim 5, wherein the C-10 carbon atomhas an R configuration and the carbon atom at C-17 has an Sconfiguration.
 9. The method of any of claims 4-8, wherein P₁ and P₂each are hydrogen atoms, or a pharmaceutically acceptable salt thereof.10. The method of claim 9, wherein Z is a carboxylic acid or acarboxylic ester, or a pharmaceutically acceptable salt thereof.
 11. Themethod of claim 10, wherein Z is a carboxylic acid salt.
 12. The methodof any of claims 4-8, wherein Z is COOR^(a), wherein R^(a) is hydrogen,(C1-C6) alkyl, (C3-C8) cycloalkyl, (C4-C11) cycloalkylalkyl, or (C6-C16)arylalkyl, or a pharmaceutically acceptable salt thereof.
 13. The methodof claim 1, wherein the compound has the structure:

or a pharmaceutically acceptable salt or ester thereof.